Yeast transformation methods

FIELD: biotechnologies.

SUBSTANCE: invention proposes a method for obtaining yeast library including incubation of yeast cells in a solution with 0.01-1 M of lithium acetate and 1-100 mM of dithiothreitol, obtaining a suspension containing a linearised DNA vector and a DNA insert in the ratio of 1:0.5-1:10, sorbite and CaCl2 or MgCl2 at the ratio of 4 mcg of the DNA vector to 400 mcl of 1.6×109 yeast cells/ml, and electroporation of the solution of yeast cells with a suspension at voltage of 0.5-12.5 kV/cm with capacity of 10-50 mcF.

EFFECT: invention can be used for production in yeast cells of recombinant products and creation of libraries.

15 cl, 4 dwg, 8 tbl, 9 ex

 

A related application

This application claims the priority benefits of provisional application U.S. number 61/067910, filed March 3, 2008

The scope of the invention

The invention relates to the field of transformation of yeast, the yeast cells, the libraries of yeast cells transformed thereby, and to the products in their recombinant products. More specifically, the present invention relates to the transformation of yeast by electroporation.

Background of the invention

Therapeutic antibodies, obtained from immunization of animalsin vivoor by way of the display recombinant antibodyin vitrowas successful in clinical practice, and as such confirmed that these methods are effective ways of finding new medicines. While, as a rule, expect the monoclonal antibodies obtained by immunization of animals, have a high affinity to achieve therapeutic efficacy, for the development of therapeutic antibodies upon immunization of animals required, or humanization of non-human antibodies, or access to transgenic animals expressing human antibodies. Direct selection of fully human antibodies of the pre is received libraries of antibodies by methods of display in vitro(phage, bacterial, yeast display, display of mammalian cells and ribosomal display) (Chao, G. et al. (2006) Nat. Protoc. 1:755-68; Gai, S. A., and Wittrup, K. D. (2007) Curr. Opin. Struct. Biol. 17:467-73; Griffiths, A. D. et al. (1994) EMBO J. 13:3245-60; He, M. and Khan, F. (2005) Expert Rev. Proteomics 2:421-30; Hoogenboom, H. R. (2002) Methods Mol. Biol. 178:1-37; Hoogenboom, H. R. (2005) Nat. Biotechnol. 23:1105-16) offers a valuable parallel way and may represent the best alternative in cases where the antigen target may not cause a productive immune responsein vivo.

Designed libraries of human antibodies for the display of full-size antibodies or fragments of antibodies, such as Fab, scFv and dAb. Libraries, as a rule, design or reflection and the transfer of the diversity of the antibody repertoire of B-cells in the library with the additional introduction of synthetic diversity or without (Hoet, R. M. et al. (2005) Nat. Biotechnol. 23:344-8), or by synthetic random distribution of residues of the CDR in a limited frame of human antibodies (Rothe, C. et al. (2008) J. Mol. Biol. 376:1182-200). Because of the heavy and light chains of antibodies are amplified separately and was reformatted into a format library display, the new Association between VH and VL are formed during this process. While this rearrangement of VH-VL allows you to create new antigennegative areas, it is also very significantly increased the AET theoretical diversity of the library. Developed several strategies for designing better and more productive libraries of antibodies by reducing theoretical diversity of the library and the actual size of the libraries required for efficient sampling from the library. These methods include intelligent design of synthetic or semi-synthetic libraries (Rothe, C. et al. (2008) J Mol Biol. 376:1182-200) and immune (Hoet, R. M. et al. (2005) Nat. Biotechnol. 23:344-8) or pseudomonic (Lee, H. W. et al. (2006) Biochem. Biophys. Res. Commun. 346:896-903) libraries derived from less diverse, but potentially more reactive repertoires of B-cells. Although these methods can be effective for a significant reduction in theoretical diversity of the library for several or even many orders of magnitude, the need for larger libraries and methods for their preparation will always complement the design of libraries to increase the chances of identification found antibodies regardless of their rarity.

The availability of funds to obtain libraries of nucleic acids and produced them in recombinant products, such as pharmaceutical proteins in eukaryotic systems such as yeast, provides significant advantages compared to the use of prokaryotic systems, such asE. coli. Yeast, as a rule, you shall asiati to a higher density of cells, than bacteria and can easily be adapted for processing continuous fermentation. However, the development of species of yeast as a systems owner/vector to obtain a recombinant products and libraries are severely hampered by lack of knowledge about the conditions of transformation and suitable means for the stable introduction of foreign nucleic acids in yeast cells-hosts.

Among the various electrical and biological parameters that facilitate electrotransformation cells, there is an adsorption of DNA on the surface of cells. Varying electric fields of low intensity also stimulate DNA transfer in bacteriaE. colipresumably, by electrical stimulation of permease DNA. Accumulated evidence prevailing electrodiffusion or electrophoretic effect on gene transfer by electroporation for polyelectrolyte DNA. Published also relieved electroosmotic effects and protrusion of the membranes by electroporation.

The application of an electric field through the membrane of the yeast cells leads to the formation of transient pores, which are critical for the process of electroporation. A signal generator for electroporation provides a voltage (in kV), passing through the gap (in cm) between the electrodes. This potential difference is definitely the ing, what is called the strength of the electric field, where E is equivalent kV/cm, Each cell has its own critical field strength for optimal electroporation. This is due to the size of the cell, membrane structure and individual characteristics of the cell wall. For example, for mammalian cells, usually need between 0.5 and 5.0 kV/cm to cell death and/or the passage of electroporation. Typically, the required strength of the field varies in inverse relation to the size of the cell.

Methods of transformation

1. Transformation by electroporation

Becker et al. (Methods in Enzymology 194: 182-187 (1991)) describe how the transformation of the yeastSaccharomyces cerevisiae(S. cerevisiae). Becker describes the transformation of spheroplasts.

Faber et al. (Curr. Genet. 25: 305-310 (1994)) describe how the transformation of the methylotrophic yeastHansenula polymorpha. Faber also applied the method toPichia methanolica.

Helmuth et al. (Analytical Biochem. 293:149-152 (2001)) describe the increase in efficiency transformation of yeast by a combination of pre-treatments as LiAc and DTT.

Kasutske et al. (Yeast 8: 691-697 (1992)) describe the electro-transformation of intact cells ofCandida maltosavarious homologous vector plasmids.

Meilhoc et al. (Bio/Technology 8: 223-227 (1990)) describe a system transformation with the use of intact yeast cellsS. cerevisiaeand pulse is elektricheskogo field.

Neumann et al. (1996) Biophys. J. (1996) 71:868-877 describe the kinetics of the transformation of yeast cells by electroporation and mediated calcium adsorption of DNA.

Piredda et al. (Yeast 10: 1601-1612 (1994)) describes a system transformation for yeastYamadazyma (Pichia) ohmeri.

Scorer et al. (Bio/Technology 12: 181-184 (1994)) describe the vectorsP. pastoristo allow rapid selection with G418 rare transformants with a large number of copies for expression inPichia pastorisusing systems such as electroporation and transformation spheroplasts.

Sherman et al. (Laboratory Course Manual for Methods in Yeast Genetics, pages 91 to 102, Cold Spring Harbor Laboratory (1986)) describe the transformation mutants of the yeast LEU2 and HIS3.

Suga and Hatakeyama (Curr. Genet. 43:206-211 (2003)) describe a method of freezing to obtain competent cells prior to electroporation by adding calcium.

Thompson et al. (Yeast 14:565-571 (1998)) describe the preparation of yeast cells, such asS. cerevisiaeandCandida albicansfor transformation by electroporation.

Yang et al. (Applied and Environmental Microbiology 60(12): 4245-4254 (1994)) describe electroporationPichia stipitisbased on their URA3 gene and homologous offline can replicate sequence ARS2.

In U.S. patent No. 5716808 from Raymond describes how to get the cellsPichia methanolicacontaining alien design DNA using electroporation and methods of production of foreign peptides in cellsPichia methanolica .

In U.S. patent No. 7009045 from Abbas described the transformation plaminogen yeast,Pichia guilliermondii yeastandCandida famataby electroporation, and through the transformation of spheroplasts.

2. Transformation through education spheroplasts

Becker et al. (Methods in Enzymology 194: 182-187 (1991)) describe how the transformation of the yeastS. cerevisiaeas well as the transformation of spheroplasts.

Scorer et al. (Bio/Technology 12: 181-184 (1994)) describe the vectorsP. pastoristo allow rapid selection with G418 rare transformants with a large number of copies for expression inPichia pastorisusing systems such as electroporation and transformation spheroplasts.

In U.S. patent No. 4808537 from Stroman et al. describes the isolation and cloning induced by methanol gene ofPichia pastorisand regulatory region, applicable to regulation by methanol expression of heterologous genes using transformation spheroplasts.

In U.S. patent No. 4837148 from Cregg et al. the described sequence for Autonomous replication capable of maintaining the plasmid in the form of extrachromosomal elements in strains of hostsPichia. In addition, the patent describes the design, including DNA sequences, as well as the transformed organisms obtained through education spheroplasts, and provided the means of obtaining the DNA sequences of the constructs according to the invention, as well as how the selection of sequences from any source.

In U.S. patent No. 4855231 from Stroman et al. described DNA sequences responsible for the presence of methanol, not repressed by catabolites sources of carbon and starvation for carbon sources. In patent '231 shows the transformation of spheroplastsPichia pastoris.

In U.S. patent No. 4879231 from Stroman et al. describes how transformation spheroplasts yeast, such asPichia pastoris.

In U.S. patent No. 4882279 from Cregg et al. describes how transformation spheroplasts for yeast of the genusPichiafor example,Pichia pastoris.

U.S. patent No. 5135868 from Cregg relates to a method sitespecifically genomic modification of yeast of the genusPichia. In patent '868 use the method of transformation spheroplasts.

U.S. patent No. 5268273 from Buckholz relates to a method of transformation of spheroplastsPichia pastoris.

U.S. patent No. 5736383 from Raymond relates to a method of transformation of yeast strains of the genusPichiain particular,Pichia methanolica. In addition, the patent '383 relates to a method of transformation of spheroplasts yeast of the genusPichiaas well as a way of transformation by electroporation.

3. Other systems transformation

Kunze et al. (Current Genetics 9(3): 205-209 (1985)) describe how the transformationS. cerevisiae,Candida maltosaandPichia guilliermondii yeast G266the plasmid pYe(ARG4)411, containing the gene ARG4S. cerevisiaein the purposes of pBR322. Kunze used CaCl2in the process of transformation.

Kunze et al. (J. Basic Environ. 25(2): 141-144 (1985)) describe how the transformation of industrially important yeastCandida maltosaandPichia guilliermondii yeast G266using CaCl2.

Kunze et al. (Acta Biotechnol. 6(1): 28 (1986)) describe the transformation of industrially important yeastCandida maltosaandPichia guilliermondii yeast.

Neistat et al. (Mol. Ge. Mikrobiol. Virusol. 12: 19-23 (1986))(Only abstract) describe the transformation of the yeastHansenula polymorpha,Pichia guilliermondii yeast,Williopsis saturnusthe plasmid carrying the gene ADE2S. cerevisiae. The method of transformation is not described.

In U.S. patent No. 4929555 from Cregg et al. describes a method for whole cell methylotrophic species of the genusPichiacompetent for transformation by DNA and the method of transformation with the DNA of whole cells of these species, in particular,Pichia pastoris.

In U.S. patent No. 5231007 from Heefner et al. described is a method of obtaining and allocating highly plaminogen strains ofCandida famata,producing outputs Riboflavin approximately 7,0-7,5 grams per liter for 6 days. The method includes the combination of repetitive steps of mutagenesis and fusion of protoplasts, conducted for the original strain and derived from it strains, which are taken after each stage according to the screening Protocol.

4. Vectors, elements of ARS and libraries of genes

Clyne, R. K. et al. (EMBO J. 14(24): 6348-6357 (1995)refers to the thin structure of the resultant analysis ARS1, item ARS fission yeastSchizosaccharomyces pombe. Characterization of a series of mutations, with a female deletions showed that the minimum DNA fragment containing ARS1, is great, because not a single fragment below 650 BP was not retained significant activity ARS.

Liauta-Teglivets, O. et al. (Yeast 11(10): 945-952 (1995)) describe the cloning of the structural gene GTP-cyclohydrolase involved in the biosynthesis of Riboflavin, from genomic librariesPichia guilliermondii yeast.

Cannon, R. D. et al. (Mol. Gen. Genet. 221(2): 210-218 (1990)) describe the isolation and nucleotide sequence of the Autonomous element can replicate sequence (ARS), function inCandida albicansandS. cerevisiae.

Takagi, M. et al. (J. Bacteriol. 167(2): 551-555 (1986)) describe the design of the system vector-host inCandida maltosausing plot ARS, isolated from their genome.

Pla, J. et al. (Gene 165(1): 115-120(1995)refers to DNA fragments ARS2 and ARS3Candida albicanswith the activity of Autonomous replication, contributing, as shown, reintegrative genetic transformation asCandida albicansandS. cerevisiae.

In U.S. patent No. 5212087 from Fournier et al. the described sequence of ARS, which is effective inYarrowia lipolyticaas well as the plasmids carrying these sequences.

In U.S. patent No. 5665600 from Hagenson et al. described linear plasmidsPichia pastorisDNA fragments containing the sequence of ARS. The Pat is the '600 used system transformation spheroplasts, as described by Cregg et al in U.S. patent No. 4929555.

In U.S. patent No. 4837148 from Cregg et al. described can replicate autonomously sequence, is able to maintain the plasmid in the form of extrachromosomal elements in strains of hostsPichia. In addition, the patent '148 refers to the structures that contain DNA sequences, as well as transformed their organisms. In addition, the patent relates to methods for DNA sequences and structures according to the invention, as well as to methods of allocation sequences from any source.

Chao et al. (Nature Protocols, 1(2):755-768 (2006)) describes how the transformation of yeast cells by electroporation and the maximum size of the library 5 × 107using 5 μg of the DNA insert and 1 µg DNA of the vector.

The above methods and descriptions, while allow you to achieve higher efficiency of transformation, are still time-consuming and take considerable time and repeated efforts to accumulate many small libraries in the size range 106-107the larger and the combined library with a size in the size range 108-109.

For yeast libraries do not reach the size or efficiency achieved for phage libraries. Review Hoogenboom in 2005 (Nature Biotech., 23(9):1105-1116), the typical maximum size of the R ragovoy library is 10 10-1011, while the size of a typical yeast library is 107(much less than the size achievable with different display technologies (Hoogenboom, H. R. (2002) Methods Mol. Biol. 178:1-37; Hoogenboom, H. R. (2005) Nat. Biotechnol. 23: 1105-16), although previously published sizes of libraries in the 109(Hoet, R. M. et al. (2005) Nat. Biotechnol. 23:344-8; Lipovsek, D. et al. (2007) J. Mol. Biol. 368:1024-41; Segal, L. et al. (2007) Bioinformatics 23:i440-9. Feldhaus et al. (Nature Biotech., 21: 163-170 (2003)) showed that the library of 1.5 × 109can be constructed through careful repetition of the transformation and unity then transformed libraries. Although recent progress in methods of electroporation (see Chao, Nature Protocols 1(2):755-768 (2006)) has made it possible to achieve the maximum size of the yeast library 5 × 107for a single transformation, the authors of the present invention discovered that by way Chao yeast usually transform with much lower transformation efficiency. Still a correct statement that these dimensions yeast libraries achieved to date, is still significantly below those that can be achieved in a conventional manner by means of libraries of phage display size 1010-1011.

The selection of libraries of yeast display, using magnetic beads and fluorescence activated sortiront the cells, provides an efficient and sensitive method for the enrichment of members that specifically bind antigens of the target, in particular, due to their compatibility with fluorescence activated sorting of cells (FACS). The advantage of this power of selection, however, constrained by limited size of a typical libraries yeast display due to the low efficiency transformation of yeast cells. If the method of selection of yeast display could be merged with larger libraries of antibodies, similar to the libraries obtained for phage display (approximately 1010), the way yeast display would be an extremely effective tool for the detection of antibodies.

Thus, there is a need for efficient ways of producing libraries of proteins, for example, libraries of antibodies using yeast.

Summary of the invention

The invention relates to a highly efficient and rapid methods of transformation of yeast cells, for example, to obtain libraries of yeast cells by up to 2 × 1010. The methods according to the invention make it possible to achieve outputs in the scale of phage libraries. The methods according to the invention remove a significant bottleneck using yeast display as a practical tool the La achieve much more diversity, previously unexplored, and the size of the library at present is limited only by the size of the yeast culture, which can be grown in laboratories.

Many components and conditions, including the use of CaCl2, MgCl2, sucrose, sorbitol, lithium acetate, dithiothreitol, voltage electroporation, the load DNA and cell volume, tested or was titrated to determine the best combination. By applying this newly developed method, a library of antibodies 2×1010constructed from RNA spleen human being for one day with a typical efficiency of transformation of 1-1,5×108transformed in micrograms DNA vector. The sequence analysis confirmed a diverse representation of the germline sequences of the antibodies in the library of antibodies non-immune spleen person. By conducting a pilot selections from the library of the authors of the present invention identified human antibodies against TNF-α and IL-18 with low nanomolar affiniscape, showing the efficiency of this library.

In one aspect the invention relates to methods for yeast library by electroporation of yeast cells, where the method includes the stages of (a) obtaining a suspension containing the vectors of the nucleic acid, cells of yeast, sorbitol and CaCl2 ; and (b) electroporation of suspension of 0.5 kV/cm to more than 12.5 kV/cm with a capacity of from about 10 to about 50 μf. In one of the embodiments the invention relates to methods for transforming yeast with DNA to obtain a library, where the method includes the stages of (a) culturing yeast cells to OD600from about 1.0 to about 2; (b) washing the yeast cells in the same volume of cold water; (c) washing the yeast cells in the same volume of cold 1 M sorbitol/1 mm CaCl2; (d) incubation of yeast cells in one volume 30°C 0.1 M LiAc/10mm DTT; (e) washing the yeast cells in the second volume of cold 1 M sorbitol /1 mm CaCl2; (f) resuspendable of yeast cells in the third volume of cold 1M sorbitol/1 mm CaCl2for the formation of a suspension of yeast cells for electroporation; (g) adding one volume of cell suspension for electroporation for DNA vector and DNA insert for forming a suspension of yeast cells - DNA for electroporation; and (h) electroporation of suspension of yeast cells - DNA for electroporation at a voltage between about 2.5 kV/cm and approximately a 12.5 kV/cm in a ditch with a clearance of 0.2 see

The ratio of DNA vectors for DNA insert is in the range from about 1:0.5 to about 1:10, for example, 1:0,5, 1:1; 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In one embodiment, implementation, PR is approximately 1 µg DNA vector and approximately 1 μg of the DNA insert used for the reaction. In another embodiment, about 1 μg DNA vector and approximately 2 μg of the DNA insert precipitious. In another embodiment, about 1 μg DNA vector and approximately 3 µg DNA insertion precipitious. In another embodiment, about 1 μg DNA vector and approximately 4 µg DNA insertion precipitious. In another embodiment, about 1 μg DNA vector and approximately 5 μg of the DNA insert precipitious.

In one embodiment, the implementation of a cell suspension contains from about 50 to about 400 μl of yeast cells, for example, approximately 50, 100, 150, 200, 250, 300, 350, 400 μl of yeast cells.

In one embodiment, the implementation of the suspension of yeast cells contains from about 1 to about 10 × 109yeast cells/ml, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10×109yeast cells/ml

In one embodiment, the implementation of the force field used for electroporation of yeast cells, ranged from about 0.5 kV/cm to approximately a 12.5 kV/cm, for example, about 0.5, about 1.0, the approximately 2.0, about 2.5, approximately 3.0, about 3.5, will bring the flax 4,0, about 4.5, about 5.0 and about 5.5 to approximately of 6.0, about 6.5, about to 7.0, about 7.5, about to 8.0, about 8.5, about to 9.0, about 9.5, about 10.0 to approximately 10,5, approximately 11,0, approximately 11,5, approximately 12,0, around 12.5, approximately 13,0, approximately 13,5, approximately 14,0, approximately 14.5, or approximately at 15.0 kV/see

In one of the embodiments spend electroporation of yeast cells with the capacity from approximately 10 to approximately 50 microfarads, for example, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50.

In one of the embodiments of the yeast cells are suspended in from about 0.1 to about 10 M sorbitol and from about 0.1 to about 10 mm CaCl2or MgCl2,for example, approximately 0.1, approximately 0.25, about 0.5, about 0.75 to approximately 1.0 to approximately 2.0 to approximately 3.0 is approximately 4.0 to approximately 5.0 to approximately 6.0 and approximately 7,0, approximately 8.0 to approximately 9,0, or approximately 10.0 M sorbitol, or, for example, approximately 0.1, approximately 0.25, about 0.5, about 0.75, approx the positive 1,0, approximately 2.0, approximately 3.0, approximately 4.0 to approximately 5.0 to approximately 6.0 and approximately 7,0, approximately 8.0 to approximately 9,0 or approximately 10.0 mm CaCl2or MgCl2.

In one of the embodiments of the yeast cells incubated in from approximately 0.01 to approximately 1.0 M LiAc, for example, about 0.01, about 0.02 and about 0.03, roughly 0.04, about 0.05, about 0.06 to approximately 0.07 to approximately 0.08 to approximately 0,09, about 0.1, about 0.2, about 0.3, and about 0.4, about 0.5, about 0.6 to, approximately 0.7, about 0.8, about 0.9 to or approximately 1.0 M LiAc, and/or from about 1 to about 100 mm DTT, for example, about 1, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mm DTT.

In a specific embodiment, the invention relates to methods for transforming yeast with DNA to obtain a library, where the method includes one or more of the stages (a) culturing yeast cells during the night to OD600from approximately 1.0 to approximately 2.0; (b) washing the yeast cells in water; (c) washing the cells draw the Jay in the solution, contains sorbitol and CaCl2; (d) incubation of yeast cells in a solution containing LiAc and DTT; (e) washing the yeast cells in a solution containing sorbitol and CaCl2; (f) resuspendable of yeast cells in the same volume of a solution containing sorbitol and CaCl2to form a suspension of yeast cells for electroporation; (g) adding one volume of cell suspension for electroporation for DNA vector and DNA insert for forming a suspension of yeast cells - DNA for electroporation; and (h) electroporation of suspension of yeast cells - DNA for electroporation at a voltage between about 2.5 kV/cm and approximately a 12.5 kV/cm in a ditch with a clearance of 0.2 see

In another specific embodiment, the invention relates to methods for transforming yeast with DNA, where the method includes two or more stages of: (a) minimize the amount of DNA in the vector and insert in a ratio of about 1:0.5 to about 1:10 by precipitation of the DNA in the precipitate, followed by resuspending a minimum; (b) culturing the yeast cells from the colony to the appropriate state growth through (i) inoculation of the first volume of medium colony of yeast from a Cup for cultivation or part of a growing yeast culture; (ii) growing the yeast cells during the night when 30°C; (iii) inoculation of the second volume of medium tile is AMI from stage (ii) and growing the cells at 30°C, until they reach OD600from about 1.3 to about 1,6; (c) deposition of yeast cells by centrifugation; (d) washing the yeast cells with cold water; (e) washing the yeast cells cold 1M sorbitol/1 mm CaCl2; (f) resuspendable of yeast cells in 0.1 M LiAc/10 mm DTT; (g) incubating the yeast cells at 30°C at 250 rpm for 30 minutes; (h) washing the yeast cells with 1 M sorbitol/1 mm CaCl2; (i) resuspendable of yeast cells in 1 M sorbitol/1 mm CaCl2for the formation of a suspension of yeast cells for electroporation; (j) adding a portion of a suspension of yeast cells for electroporation to the precipitate DNA for the formation of a suspension of yeast cells - DNA for electroporation; (k) electroporation of suspension of yeast cells - DNA for electroporation in the range from about 0.5 kV to about 2,5 kV and a capacitance of 25 μf; (1) adding a mixture of 1:1 to 1 M sorbitol/YPD (final concentration: 0.5 M sorbitol and 0.5 × YPD) subjected to electroporation suspension of yeast cells - DNA for electroporation and incubation at 30°C within 1 hour; and (m) deposition of yeast cells and resuspendable in 10 ml of 1 M sorbitol.

Brief description of figures

The above and other objectives, features and advantages of the present invention, as well as the actual invention can be more fully understood from the following description of predpochtitel the different embodiments when read together with the accompanying figures:

The figure 1 shows the graphical representation of the data in table 1, which represents the expense of the colonies as a function of vector: insert and voltage 2,5 kV/cm to 12,5 kV/see

The figure 2 shows the graphical representation of the data in table 3, which represents the expense of the colonies as a function of vector: insert and voltage of 2.5 kV/cm to 12,5 kV/see

Figure 3 shows that the efficiency of electroporation significantly improved by increasing the volume of the cell.

Figure 4 shows that the increased load DNA and high voltage, but not the ratio of vector and insert are critical for maximum efficiency of transformation.

Detailed description of the invention

The method is highly efficient electroporation forSaccharomyces cerevisiaedeveloped through testing and a combination of several previously identified conditions (Chao, G. et al. (2006) Nat. Protoc. 1:755-68; Becker, D. M. and Guarente, L. (1991) Methods Enzymol. 194:182-7; Helmuth, M. et al. (2001) Anal. Biochem. 293:149-52; Neumann, E. et al. (1996) Biophys. J. 71:868-77; Simon, J. R. (1993) Methods Enzymol. 217:478-83; Suga, M. and Hatakeyama, T. (2003) Curr. Genet. 43:206-11; Thompson, J. R. et al. (1998) Yeast 14:565-71. They include a combination of lithium acetate (LiAc) and dithiothreitol (DTT) as a modifier of cell means, both of which are used to increase the frequency transformation of yeast (Helmuth, M. et al. (2001) Anal. Biochem. 293: 149-52; Thompson, J. R. et al. (1998) Yeast 14:565-71; Gietz, R. D. t al. (1995) Yeast 11:355-60), and the inclusion of sorbitol and calcium chloride (Becker, D. M. and Guarente, L. (1991) Methods Enzymol. 194:182-7; Helmuth, M. et al. (2001) Anal. Biochem. 293:149-52; Neumann, E. et al. (1996) Biophys. J. 71:868-77) for electroporation buffer.

Definitions:

The term "expressing vector" refers to a design DNA that contains the plot of Autonomous replication (ARS), the site of transcription initiation and at least one structural gene that encodes a protein that is subject expression in the body of the host. Plot replication, or the origin of replication is any DNA sequence that controls the replication cloning and expressing vectors. Expressing the vector typically contains suitable control area, such as one or more enhancers and/or promoters, suppressors and/or silencers and terminators, which controls the expression of the protein in yeast hosts. Expressing the vectors of the present invention may contain a selective marker, containing essential gene, as described herein. Expressing the vector also, optionally, contain other selective markers, widely available and well known to specialists in this field. Expressing the vectors represent one type of vector. Vectors can, optionally, contain one or more sequences(elements) ARS from one or more strains of yeast.

The term "functionally linked" means that the DNA fragments are arranged so that they function in cooperation for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the coding segment to the terminator.

The term "transformation" means introducing DNA or other nucleic acids into the cell by the host yeast-recipient that changes the genotype.

The term "transformant"or "transformed cell" means a cell of the host yeast-recipient, which was subjected to transformation, and his posterity.

Vectors that are applicable in the methods of electroporation according to the invention include a vector of pYD, and any other vectors and their derivatives designs that can be propagated in yeast cells, or nucleic acids in General. Expressing vector of the present invention can be based on any type of vector, as the vector can be transformed to transliterate or transducible yeast cell host. In a preferred embodiment expressing vector based on a yeast plasmid, especially the plasmids fromS. cerevisiae. After transformation of yeast cells exogenous DNA encoding a sequence library, is absorbed by the cells and then expressed in transformed cells.

More preferably, ek is preserue the vector may be a Shuttle vector for yeast-bacteria, which can be propagated either inE. colior in yeast (Struhl, et al. (1979) Proc. Natl. Acad. Sci. 76:1035-1039). The inclusion of the DNA sequences of plasmidsE. colisuch as pBR322, facilitates quantitative obtaining DNA vector inΕ. coliand thus, the effective transformation of yeast.

Types of yeast plasmid vectors that can serve as a Shuttle, can be a vector can replicate or integrating vector. Can replicate the vector is a yeast vector, is able to mediate their own maintenance, independently from the chromosomal DNA of yeast due to the presence of functional origin of DNA replication. Integrating the vector of recombination depends on the chromosomal DNA to facilitate replication and thus prolonged maintenance of the recombinant DNA in the cell host. Replicating vector may be a based on plasmid 2 micron vector, in which the origin of DNA replication comes from endogenous 2 micron plasmid of yeast. An alternative that can replicate the vector may be an autonomously can replicate (ARS) vector, in which "seemingly" the origin of replication is from chromosomal DNA of yeast. Optional, can replicate the vector may be a centromeric (CΕN) plasmid, carrying, in addition to one of you who ukazannyh of original DNA replication the sequence of the chromosomal DNA of yeast, a carrier, as is known, centromere.

Yeast cells can be transformed with vectors in a closed ring form or in linear form. Transformation of yeast integrating vectors, albeit with hereditary stability, may not be effective when the vector is present in a closed ring form (for example, only approximately 1-10 transformants on ug DNA). Linearized vectors, with free ends, which are localized in the DNA sequences homologous to the chromosomal DNA of yeast, transforming yeast with a higher efficiency (100-1000 times), and found that the transforming DNA, as a rule, integrates in the sequence homologous to the site of cleavage. Thus, by splitting the DNA vector of the appropriate restriction endonuclease, you can increase transformation efficiency and to target the site of integration into the chromosome. Integrative transformation can be applicable for genetic modification of yeast, provided that the transformation efficiency is quite high and the sequence of the DNA target site for integration lies within a region that does not violate the genes required for the metabolism of the host cell.

Plasmids with ARS, with a high number of copies (PR is approximately 20-50 copies per cell), tend to be the most unstable and lost with a frequency of more than 10% per generation. However, the stability of the plasmids with ARS can be increased by the accession of centromere; centromeric plasmids present in 1 or 2 copies per cell and only lost about 1% per generation. Non-limiting examples of proteins for expression in yeast cells, hosts using electroporation according to the invention include any genes of interest, including antibodies and antibody fragments, hormones, cytokines and lymphokines, receptors, adhesion molecules and enzymes.

The yeast strains that can transform the way electroporation according to the invention include species of yeast of the genusSaccharomycessuch asSaccharomyces cerevisiaeand kind ofSchizosaccharomycessuch asSchizosaccharomyces Pombe. In one embodiment, the yeast cells are diploid yeast cells. Alternatively, yeast cells are haploid cells, such as "a" and "α" strain haploid yeast cells.

The invention relates to methods of transformation of yeast cells, including electroporation of cell suspension containing yeast together with one or more structures of nucleic acids. Transformation of yeast cells may lead to anything from an individual clone to is opulatio of yeast cells (i.e. yeast libraries or libraries)that can be used for screening (a) peptide(peptides) or protein(proteins)exposed on the surface of yeast cells by attaching to a protein on the surface of yeast or binding by specific covalent bond or non-covalent interactions with proteins or other components on the surface of yeast cells; (b) peptide(peptides) or protein(proteins)expressed inside the cells; or (c) peptide(peptides) or protein(proteins)secreted in the extracellular space, such as culture medium or accumulated on solid surfaces. Such yeast library may be convenient to use for many applications, for screening or characterization of the interactions of the peptide(peptides) or protein(proteins) with another protein, peptide, DNA, RNA, or other chemical substances, which can be introduced into yeast cells or add exogenous. Specific examples of this found in a yeast display, yeast two-hybrid system yeast trihybrid system etc.

The invention relates to a method of transformation of yeast cells, including electroporation of cell suspension containing yeast together with one or more structures of nucleic acids containing one or more regulate the data sequences and one or more genes or gene fragments, using one or more of resistance, field strength and pulse duration sufficient to transform yeast cells.

In one embodiment, the implementation of the field strength is from about 2.5 kV/cm to approximately a 12.5 kV/see In particular embodiments, the implementation of the field strength is approximately 0.5 kV/cm, approximately 1.0 kV/cm, about 1.5 kV/cm, approximately 2.0 kV/cm, or about 2.5 kV/cm For these values take into account that the cuvette for electroporation has a clearance of 0.2 see More high power fields are possible, but their viability largely depends on the development machine that can deliver a stronger impulse.

In one of the embodiments, the pulse duration is from about 3 milliseconds to about 10 milliseconds. In a specific embodiment, the pulse duration is approximately 4.8 milliseconds.

Treatment of cells by means of electroporation according to the invention is carried out by application of an electric field to the suspension of yeast cells between a pair of electrodes. Force field is necessary, it is advisable accurately adjusted so that the electroporation of cells occurred without damage to cells or with minimal cell damage. You can then measure the distance between the elect is the od and then a suitable voltage according to the formula E=V/d can be applied to the electrodes (E=strength of the electric field in V/cm; V=voltage in volts; and d=distance in cm).

The pulse generators to implement the methods described herein, are available and have been available on the market for several years. One suitable signal generator is a Gene Pulser II (Bio-Rad Laboratories, Inc., Hercules, CA). A typical circuit consists of the Gene Pulser II, attached to the optional amplifier capacity and additional controller pulse.

Additional models for electroporation are commercially available, including the Gene Pulser MXcell or eukaryotic system Gene Pulser x cell (Bio-Rad), tame the electroporator CelljecT Pro (Thermo Scientific), system for electroporation Multiporator and other systems for electroporation Eppendorf® (Eppendorf, North America), generators for electroporation ECM and 96-well system for electroporation BTX®HT (BTX, Harvard apparatus), all of which are suitable devices for electroporation of yeast cells using conditions described here.

Electroporation is used in the present invention to facilitate the introduction of DNA into yeast cells. Electroporation is a method using a pulsed electric field to provide temporary permeability of cell membranes, allowing macromolecules, such as DNA, to pass into the cells. However, the actual mechanism by which DNA is transferred to glue the key, not entirely clear. For transformationCandida famatafor example, electroporation is unexpectedly effective when cells are exposed to experimental fading pulsed electric field having a field strength from about 10 to about 13 kV/cm and a resistance value of approximately R5 (129 Ohms), and a constant time approximately 4 to 5 MS. Typically, the resistance and capacitance or present, or may be selected by the user depending on the selected equipment for electroporation. In any case, the hardware configuration is set according to the manufacturer's instructions to ensure appropriate force field and parameters of decay.

In addition, the invention relates to efficient methods of transformation of yeast, providing a high level of expression of any one or more of the desired endogenous (i.e., naturally existing within this yeast cells) or heterologous genes. In addition, the methods according to the invention relate to a method for libraries, for example, expressing antibodies or chimeras or fragments.

One programme of action, expressing vectors carrying genes of interest, can be transformed cell hosts of yeast by electroporation to obtain a separate CL is at or library, consisting of many transformed cells expressing intracellular proteins (e.g., nuclear or cytoplasmic proteins), membrane proteins (for example, passing through the membrane proteins or membrane proteins), or secreted proteins. You can use the transformed cells or library for protein purification, study of the functions of proteins, identification of protein-protein interactions or to identify new binding protein substances or character interaction. An important remark is the possibility to obtain very large yeast library, exhibiting or expressing antibodies and antibody fragments. The library can be subjected to selection by antigen targets to identify antibodies that bind with selective antigens.

Since the transformed yeast tend to lose artificially constructed plasmids, is the predominant use of the culture medium, such that pressure positive selection on them. When the strain is auxotrophic mutant vital metabolite, and when used vector plasmid contains a marker gene capable of restoring prototroph strain, for example, gene LEU2 mentioned above, this selective pressure can be applied with the exception of Metabo is it from the culture medium. There are other means to obtain the same result, and they can also be used for carrying out the invention in practice.

Depending on the nature of interest structural gene product or expression product may remain in the cytoplasm of the yeast host cell or secretariats. Discovered that not only proteins, which remain in the cell, but also those that are secreted are soluble. When the product or expression product is designed to remain in a yeast cell host, as a rule, it may be desirable to have the inducible region of transcription initiation, so that while the transformant will not reach high density, expression or production of the desired product is present in a small amount or is not present. After a time sufficient for expression of the product or expression product, cells can be distinguished by using common methods, for example, by centrifugation, lysis, and select the desired product. Depending on the nature and use of the product, the lysate can be subjected to various purification methods such as chromatography, electrophoresis, extraction with solvent, crystallization, dialysis, ultrafiltration or the like Methods of chromatography include as non-limiting examples of gas chromatography, HPLC, column items is matography, ion-exchange chromatography and other methods of chromatography, known to specialists in this field. The degree of purity can vary from approximately 50% to 90% or higher, preferably up to approximately 100%.

Alternatively, the expression product or interested in the product can secrete into the culture medium and to produce on a continuous basis, where the environment is partially selected, the desired product is extracted with, for example, a column or affinity chromatography, ultrafiltration, precipitation or the like, and used the environment to throw away or recycle the resumption of the necessary components. The filtrate containing the product, after ultrafiltration can be further subjected to concentration, with subsequent evaporation, followed by crystallization or by precipitation with the use of alcohol and/or setting the pH. Specialists in this area there are many possibilities of ways. When the product is subject secretion, normally you can use the constitutive region of transcription initiation, although you can use reconstitutive area.

Unless otherwise indicated, all nucleotide sequences, first described in this document was determined using automated DNA sequencing machine (such as a model 377, PE Applied Biosystems, Inc.). Thus, as izvestno this area, for any DNA sequence determined by this automated method, any nucleotide sequence determined herein may contain some errors. The nucleotide sequence determined automatically, usually at least about 90% identical, more specifically from at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more accurately determined by other means, including manual DNA sequencing are well known in this field.

Examples

Example 1: obtaining a DNA vector

Vector pYDsTev were digested with restriction enzymes SfiI and NotI for use in recombination with DNA fragments of scFv + 300 BP To improve efficiency and prevent the transformation of uncut vector, split SfiI/NotI vector was further digested BglII and HpaI (both cut inside the ballast fragment).

Cleaved DNA was purified using Sureclean from Bioline (Randolph, MA). Briefly, the mixture after restriction cleavage was mixed with an equal volume of Sureclean. Samples were incubated for 10 minutes at room temperature. Then the samples were centrifuged for 15 the minutes at 13200 rpm and the Supernatant was discarded and the precipitates were washed with 70% ethanol and 2.5-fold volume of sample. Then the samples were centrifuged for 15 minutes at 13200 rpm, the Supernatant was removed and the sediments were dried in air. Precipitation resuspendable in 5-10 μl of TE pH 8.

Example 2: Obtaining DNA insert

The DNA insert was obtained using PCR amplification of specific insert with the addition of the insert is sufficient 5' and 3' vector sequence (for example, approximately 300 base pairs) for the effective conduct of homologous recombination.

Designed primers for the constant region of immunoglobulin (table 2), hybridization with immunoglobulins IgM and IgG (IgM cDNA/G) or IgK (cDNA IgK) for the synthesis of 1thchain cDNA. Specifically, cDNA IgM/G the primers used were FcgRev1, 2, and 3 and FcmRev1, 2 and 3. For the synthesis of 1thchain cDNA IgK used Ckrev1, 2 and 3.

The cDNA synthesis reaction was performed using a kit for the synthesis of 1thchain from Invitrogen using the above primers.

Table 1
Illustrative PCR reaction (stage 1)
IgM/G cDNAIgK cDNA
20 ál of 50 ng/μl of the poly RNA spleen, CloneTech10 μl 50 ng/μl of the poly RNA spleen CloneTech
DNA DNA
3 300 ál ng/ál FcgRev 11,5 300 ál ng/ál CkRev 1
3 300 ál ng/ál FcgRev 21,5 300 ál ng/ál CkRev 2
3 300 ál ng/ál FcgRev 31,5 300 ál ng/ál CkRev 3
3 300 ál ng/ál FcmRev 110 ál of dNTP (10 mm)
3 300 ál ng/ál FcmRev 275,5 ál of bidistilled water
3 300 ál ng/ál FcmRev 3total volume of 100 µl
20 μl of dNTP (10 mm)
142 μl bidistilled water
total volume of 200 ál
50 ál/reaction, the reaction at 65°C for 5', then to 4°C for 1 minute

Table 2
The oligonucleotides used in this study
The name of the oligonucleotideSequenceDescriptionSEQ D NO
Ckrev 1TCCACCTTCCACTGCk reverse primer 1SEQ ID NO: 1
Ckrev 2CAGGCACACAACA
G
Ck reverse primer 2SEQ ID NO: 2
Ckrev 3GAGTGTCACAGAG
C
Ck reverse primer 3SEQ ID NO: 3
FcGrev 1AGTTCCACGACACCIgG reverse primerSEQ ID NO: 4
FcGrev2GAAGGTGTGCACGIgG reverse primerSEQ ID NO: 5
FcGrevCCACGCTGCTGAGIgG reverse primerSEQ ID NO: 6
FcMrev 1ACTTTGCACACCACIgM reverse primerSEQ ID NO: 7
FcMrev 2TTTGTTGCCGTTGGIgM reverse primerSEQ ID NO: 8
FcMrev 3GGGAATCTCACAG
G
IgM reverse primerSEQ ID NO: 9
PYD5prevGCGCCCTGAAAATA
CAGGTTTTC
Hybridized directly at the 5'-end from the SfiI site in the vector pYD. Used in conjunction with primer 5107 to obtain the elongation of approximately 300 BP, added to the scFv to increase the efficiency of homologous recombinationSEQ ID NO: 10

Stage 2 synthesis:

45 μl of the concentrated reaction mixture (prepared starting solution containing 80 ál of buffer for RT (10×), 160 μl MgCl2,80 μl of 0.1 M DTT, 40 μl of RNAse Out), was added to each 50 μl of the cooled reaction mixture and the tubes were placed at 42°C for 2 minutes. In each reaction tube was added 5 μl of reverse transcriptase SuperscriptII and incubated at 42°C for 50 minutes, transferred to 70°C for 15 minutes, and then transferred to 4°C. each reaction mixture was added to 5µl RNase H and then incubated at 37°C for 20 minutes. Four reaction mixture for IgG/M were combined, and two of the reaction mixture for IgK together.

Example 3: Obtaining DNA IgG/M VH and IgK VL spleen person

The diversity of the immunoglobulin VH and VK person amplified using oligonucleotide primers defined in Sblattero andBradbury (1998) Immunotechnology 3:271-278.

8 × 100 μl reaction mixture for samples VH
80 ál10× reaction buffer for PCR
8 áldNTP (10 mm)
32 álMgSO4(50 mm)
8 álreverse primer for VH (20 μm)
8 álequimolar mixture of direct VH 1/2, VH4/5, VH3, and VH6 (5 μm each/total 20 μm 3'primer)
16 álcDNA IgM/G
8 álPlatinum Taq HiFi
640 álbidistilled water
100 ál/reaction mixture

8 × 100 μl reaction mixture for samples VK
80 ál10× reaction buffer for PCR
8 áldNTP (10 mm)
32 ál MgSO4(50 mm)
8 álreverse primer for VK (20 μm)
8 álequimolar mixture of VK1, VK2/4, VK3 and VK5 (5 μm each/total 20 μm 3'primer)
16 álcDNA IgK
8 álPlatinum Taq Hi Fi
640 álbidistilled water
100 ál/reaction mixture

The PCR program:

1. 94°C for 2 minutes

2. 94°C 30 seconds

3. 55°C 30 seconds

4. 68°C for 30 seconds

5. repeat stage 2 39 times

6. 68°C for 5 minutes

7. 4°C storage

The reaction mixture was purified in the gels of 1% GTG agarose. Bands of the correct size, approximately 400 BP was purified using a kit for the selection of the Qiaquick gel. 0.4 mg containing DNA agarose used for zentrifugenbau column. Samples were suirable with 50 ál EB, preheated to 55°C. Similar samples were combined and A260/280was determined using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, Delaware).

Example 4: Obtaining scFv VH/VK

scFv VH/VK was obtained by PCR with the joining of overlapping fragments (SOE) PCR. Direct VH and reverse K primers shared homology, forming linker (Gly4Ser)3connecting region VH and VL to obtain scFv molecules. The reaction mixture was prepared as follows:

24 the reaction mixture at 100 μl/reaction mixture (build VH10/VK as an example)
240 ál10x buffer for Plat Taq Hi Fi
24 áldNTP (10 mm)
96 álMgSO4(50 mm)
24 álscFv Rev (20 μm)
24 álscFv For (20 μm)
141 μlreverse VH10 (24 pmol VH/1 pmol per 100 μl reaction mixture)
72 álequimolar mixture of DNA VK1, 2, 9, and 12 (24 pmol total/0.25 pmol of each VK 100 μl reaction mixture)
24 álPlatinum Taq HiFi
1755 álbidistilled water
100 ál/reaction mixture

The PCR program

1. 94°C for 2 minutes

2. 94° for 30 seconds

3. 55°C 30 seconds

4. 68°C 1 min

5. The transition to stage 2 39x

6. 68°C for 5 minutes

7. 4°C storage

Strip scFv (approximately 800 BP) was gel purified as above for fragments of the gel with VH or VK. Similar samples were combined for use in the SOE PCR fragment pYD5'300 BP

To ensure increased efficiency of transformation of the authors of the present invention constructed fragments of the DNA insert with approximately 300 BP of homology at the 5'-end from the site of insertion of scFv (SfiI) in the vector pYD. To add this homology to scFv spleen person designed a primer, complementary to the region directly at the 5'-end of the plot SfiI pYD1sTev (pYD5prev). This primer was used in combination with 5'-primer pYD1 (5107) to obtain a fragment of about 300 BP to add to an existing scFv spleen by SOE PCR.

PCR 5107/pYD5prev
1 álpYDsTev (10 ng/µl)
10 ál10× buffer for Plat Taq Hi Fi
4 álMgSO4(50 mm)
1 áldNTP (10 mm)
1 ál5107 (20) - Rev. km)
1 álpYD5prev (20 μm)

1 álPlatinum Taq Hi Fi
80 álbidistilled water
100 ál/reaction mixture

The PCR program

1. 94°C for 2 minutes

2. 94°C 30 seconds

3. 55°C 30 seconds

4. 68°C for 30 seconds

5. The transition to stage 2 39x

6. 68°C for 5 minutes

7. 4°C storage

Strip 5107/pYD5prev (approximately 300 BP) was gel purified as above for fragments of the gel with VH or VK. Similar samples were combined for use in the SOE PCR with scFv spleen.

scFv+300 PCR (mixture for 16×100 μl reaction mixture)
162 ál10× buffer for Plat Taq Hi Fi
16,2 áldNTP (10 mm)
64,8 álMgSO4(50 mm)
16,2 ál5107 (20 μm)
16,2 álscFv For (20 μm)
32,4 ál VH/VK scFv DNA
16,2 ál5107/pYDprev5 DNA
16,2 álPlatinum Taq HiFi
1279,8 álbidistilled water
100 ál/reaction SMEs

PCR reaction was performed as for the reaction with scFv above. ScFv+300 BP (approximately 1100 BP) was gel purified as above for fragments of the gel with VH or VK. Similar samples were combined for use in electroporation reactions.

Example 5: a Way of transforming a yeast library of previous technology

The following is a method of transformation of yeast from the preceding technical field. Five ml of YPD medium inoculant colony EBY100 (svezheproseyannuyu touch the Cup with YPD and grown overnight at 30°C. 50 ml of culture inoculant in YPD medium to an optical density of 0.1 at 600 nm using a night of culture, and cells were grown at 30°C prior to the acquisition of approximately 1.3 to 1.5 at 600 nm for about 6 hours. Cells must be in the home - the average logarithmic growth phase. The use of cells in late logarithmic or stationary phase significantly reduces the efficiency of transformation. During growth to etoc DNA precipitious for transformation using PelletPaint according to the method of the manufacturer. Typically, the DNA to be placed in four cuvettes for electroporation, each with 5 mg insert 1 mg cut vector, is used to produce libraries of approximately 5 × 107. Also get control of one of the skeleton. DNA left in the form of sludge. The ratio of insert:the frame can be changed from 5:1 to 1:1 with at least 1 mg of the skeleton in a cell.

When the cells optical density of approximately 1.3 to 1.5 at 600 nm, 500 ál of Tris-DTT buffer is added to the culture and incubated in a shaker at 30°C for 15 minutes. Transformation efficiency is relatively constant for the time of incubation with DTT 10-20 minutes, but significantly reduced by incubation for 20 minutes. The precipitated cells at 2500 g for 3 minutes at 4°C and washed with 25 ml of ice-cold buffer E (10 mm Tris pH 7.5, 270 mm sucrose, 1 mm MgCl2) (for example, washed, re-precipitated and the supernatant is sucked off). The cells are again washed with 1 ml ice-cold buffer E and resuspending in buffer E in a total volume of 300 μl. Control DNA precipitation resuspended in an appropriate volume of cell suspension (50 µl per cell). Cells keep on ice. An aliquot of 50 ál resuspending mixture of cells and DNA are selected in a pre-chilled cuvette for electroporation, the cuvette for electroporation keep on ice until processing pulses.

The cell load into gene pulser and all Laut electroporation at 0.54 kV and 25 μf without a controller pulses. 1 ml of warm (30°C) environment YPD immediately added to the cuvette. Typical time constants for electroporation are in the range from about 15 MS to 40 MS without a large impact on the efficiency of transformation.

Cells are transferred from subjected to pulse ditch in 15-ml Falcon tube. Each cuvette was washed with an additional 1 ml of YPD medium to extract the remaining cells from the cell. Cells shaken for 1 hour at 30°C. the precipitated Cells at 2500 g for 5 minutes, supernatant removed and resuspended in 10 ml of medium SDCAA. Serial cultivation scatter of the Cup SDCAA to determine the transformation efficiency. Control with one skeleton must have efficiency less than 1% efficiency transformation carcass-plus-insert. Cell suspension is transferred into a bottle with 100-1000 ml of medium SDCAA plus penicillin-streptomycin (dilution 1:100) and incubated at 30°C for 24-48 hours. This method is used to obtain library 8 × 107and it corresponds to processing 7 described in table 3 with an efficiency of 0.4% compared with the optimized method according to the invention (see example 6).

Example 6: the Protocol transformation of yeast libraries from method 2 according to the invention

The invention relates to a highly efficient and rapid methods of transformation of yeast cells, for example, to obtain the library is of yeast cells by up to 2 × 10 10. Many components and conditions, including the use of CaCl2, MgCl2, sucrose, sorbitol, lithium acetate, dithiothreitol (DTT), voltage electroporation, the load DNA and cell volume, tested and was titrated to identify optimal conditions.

The method of electroporation according to the invention is a variant of the ways Suga and Hatakeyama (2003) Curr. Genet. 43:206-211; Neumann et al. (1996) Biophys. J. (1996) 71:868-877; Thompson et al. (1998) Yeast 14:565-571; Becker and Guarente (1991) Methods Enzymol. 194, 182-187; Melihon et al. (1990) Biotechnology 8:223-227; Helmuth et al. (2001) Analytical Biochem. 293:149-152; and the method presented in example 5.

Preparation of yeast cells:S. cerevisiaewere grown overnight to stationary phase. An aliquot of the culture was inoculable in 100 ml of YPD medium until reaching OD600approximately 0.3. Cells were grown until reaching OD600approximately 1.6 to collect by centrifugation. The precipitated cells were washed twice with cold water, once with 50 ml for electroporation buffer (1 M Sorbitol/1 mm CaCl2), and incubated in 20 ml of 0.1 M LiAc/10 mm DTT at 30°C for 30 minutes. Cells were again washed with 50 ml buffer for electroporation, and then suspended in 100-200 ál of buffer for electroporation to achieve a volume of 1 ml, This corresponds to approximately 1 to 10 × 109cells/ml

Electroporation:for electroporation used 100, 200, 300 or 400 µl of cell suspension in a cell and use the Ali 1, 2, 3, or 4 μg of linearized vector with the corresponding 3, 6, 9 or 12 μg DNA inserts (the ratio of vector to insert = 1:3). Cells were subjected to electroporation at 2.5 kV and 25 μf in a ditch BioRad GenePulser (the space between the electrodes is 0.2 cm). Typical time constants for electroporation were lying in the range from 3.0 to 4.5 milliseconds. After electroporation the cells are suspended in 10 ml of a mixture 1:1 1 M sorbitol:Wednesday YPD and incubated at 30°C for 1 hour. Then the cells were collected and cultured in medium SD-UT (-ura, -trp)containing 20 g/l glucose, 6.7 g/l nitrogen bases environment for yeast without amino acids, 5,4 g/l Na2HPO4, 8.6 g/l NaH2PO4H2O and 5 g/l of esamination. The number of transformants was determined by sieving 10-fold serial dilutions of transformed cells on selective cups SD UT. Referring to figure 3, after 72 hours, colonies were counted and transformation efficiency (A) was expressed as the average of the number of transformants/µg DNA vector ± standard deviation; and o (B) expressed as the average of the total number of transformants in a cell ± standard deviation.

The combination of LiAc, DTT, CaCl2and sorbitol led to the transformation efficiency 3 ± 1 × 107the transformants on ug DNA vector for 1 ág DNA vector and 100 μl of yeast cells at a density of 1.6×10 cells/ml (as determined by optical density at 600 nm, p is inima 1 OD equivalent to 10 7cells) (Figure 3). This suggests that approximately 2% of yeast cells have been successfully transformed, and the size of the library 3±1×107you can expect from one of the cuvette for electroporation. To obtain libraries even larger estimated the optimal amount of cells on a single cell. Unexpectedly, the transformation efficiency was significantly increased up to 1×108the transformants on ug DNA vector, when the volume of the cells was increased from 100 µl to 200 µl per cell. If the maximum cell volume 400 µl in a cell with a clearance of 0.2 cm, the size of the libraries, so large as 5×108could easily be obtained from a single cell using the same density and the ratio of cells to DNA (figure 3).

This method of electroporation is highly reproducible, as similar transformation efficiency was obtained two different operator and on two different electroporation (BioRad Gene Pulser™ model # 1652076 and Gene Pulser®II model # 1652108).

Example 7: Increased efficiency electroporation of S. Cerevisiae when comparing the use of LiAc, DTT, sorbitol and CaCl2

Table 3 shows the efficiency of electroporation, exit to the cuvette % efficiency and the maximum size of the library to the method of example 6, as well as variants of the method.

Table 3
ProcessingThe efficiency of electroporationa×107Output cellb×107% efficiencycThe maximum size of the libraryd
1. Combined method18,3±2,573,2±9,71002×1010
2. Without LiAc1,7±0,16,7±0,49of 1.4×109
3. Without DTT0,81±0,0173.3V±0,0748×108
4. Sorbitol/MgCl211,4±0,945,5±3,5629×109
5. Only sorbitol9±1,836±7,1498×109
6. Sucrose/CaCl20,±0,3 1,6±1,52of 3.8×108
7. Sucrose/MgCl20,09±0,010,4±0,070,48×107
aThe number of transformants on ug DNA vector
bThe total number of transformants on the reaction cuvette for electroporation
cTaking the transformation efficiency obtained by using method 100%
dTaking holding of 25 reaction cuvette for electroporation per night

The combined method is described in example 6 method of electroporation 400 µl of yeast suspension (1,6×109/ml) with 4 μg of vector and 12 μg DNA insert. For other treatments combined method followed the example 6 except for the following changes: (2) the Cells were pre-treated only DTT; (3) the Cells were pre-treated only LiAc; (4) MgCl2used instead of CaCl2. (5) CaCl2excluded from the buffer for electroporation; (6) 270 mm sucrose was used instead of 1M sorbitol; and (7) 270 mm sucrose/1 mm MgCl2 used as a buffer for electroporation.

As shown in table 2, excluding pre-treatment DTT or LiAc has led to a corresponding decrease in the efficiency of 93.3% and 85.7%. Similarly, the exclusion of sorbitol or its replacement sucrose resulted in the loss of more than 96% efficiency (table 2 and data not presented). This corresponds to previous findings that the ability of fluids to stabilize the osmotic pressure and to maintain the integrity of the membrane of the yeast after electroporation was at least partially responsible for the achievement of high efficiency of transformation (Becker, D.M. and Guarente, L. (1991) Methods Enzymol. 194:182-7; Weaver, J. .et al. (1988) FEBS Lett. 229:30-4). The presence of CaCl2during electroporation, presumably facilitating the binding of DNA with the cell membrane (Neumann, E. et al. (1996) Biophys. J. 71:868-77), apparently is the least critical parameter, because the exception only moderately reduces transformation efficiency by 30% and replace it with MgCl2leads only to a small 11% decrease.

Example 8: Effect of voltage on the efficiency of electroporation

The experiment was carried out to determine the effect of voltage on the efficiency of electroporation and to determine the best ratio of vector:insert. The experiment was performed twice. The method of example 6 assests is whether using multiple voltages in the range 0.5-2.5 kV and with several ratios of vector:insert in the range from 1:1 to 1:10 (wt./mass.), where the amount of DNA vector was maintained constant at 1 μg. All of the reaction mixture was concentrated and precipitiously using SureClean and resuspending precipitate DNA in ddH2O before adding the cell suspension, as described above.

In each experiment, the yeast cells were prepared as described above. The amount of vector/insert recorded 11.6 ál for 5 reactions.

Experiment #1

Table 4
The graph describes the number of colonies as a function of vector:insert and force fields1
2.5 kV/cm5 kV/cmof 7.5 kV/cm10 kV/cmof 12.5 kV/cm
1:10028321
1:20226170160
1:3 0184212104
1:461104362139
1:5016117968
1:100352182126
1Electroporation was performed in a cuvette with a clearance of 0.2 cm at a voltage in the range from 0.5 to 2.5 kV
Table 5
The transfection efficiency as a function of vector:insert and force fields1
The efficiency of transfectionof 7.5 kV/cm10 kV/cmof 12.5 kV/cm
1:12,00E+068,30E+072,10E+07
1:22,60E+071,70E+081,60E+08
1:38,40E+072,12E+081,04E+08
1:41,04E+083,62E+081,39E+08
1:56,10E+071,79E+086,80E+07
1:105,20E+071,82E+081,26E+08
1Electroporation was performed in a cuvette with a clearance of 0.2 cm at a voltage in the range from 0.5 to 2.5 kV

Results: Transformation were detected with a ratio of vector:insert 1:1 in the range is atragene 1.5 to 2.5; with a ratio of vector:insert 1:2 in the voltage range of 1.0 to 2.5; ratio of vector:insert 1:3 in the voltage range of 1.0 to 2.5; ratio of vector:insert 1:4 0,5-2,5; ratio of vector:insert 1:5 in the voltage range of 1.0 to 2.5; and a ratio vector:insert 1:10 in the voltage range of 1.0 to 2.5. For all ratios of vector:insert the maximum number of colonies was achieved using a voltage of 2.0 kV. Optimum conditions, apparently, was the ratio of vector:insert 1:4 and a voltage of 2.0 kV, where he reached the efficiency of transfection 3,62 × 108.

Experiment #2

Table 6
The graph describes the number of colonies as a function of vector:insert and force fields1
Data set 22.5 kV/cm5 kV/cmof 7.5 kV/cm10 kV/cm12,5 kV/cm
1:101196544
1:20045110 154
1:3013792152
1:40150136160
1:50151116163
1:10010123167
1Electroporation was performed in a cuvette with a clearance of 0.2 cm at a voltage in the range from 0.5 to 2.5 kV

Table 7
The transfection efficiency as a function of vector: insert and force fields1
The efficiency of transfectionof 7.5 kV/cm10 kV/cmof 12.5 kV/cm
1:11,90E+076,50E+07 4,40E+07
1:24,50E+071,10E+081,54E+08
1:33,70E+079,20E+071,52E+08
1:45,00E+071,36E+081,60E+08
1:55,10E+071,16E+081,63E+08
1:100,00E+001,23E+081,67E+08
1Electroporation was performed in a cuvette with a clearance of 0.2 cm at a voltage in the range from 0.5 to 2.5 kV

Results: Transformation were detected with a ratio of vector:insert 1:1 in the voltage range of 1.0 to 2.5; ratio of vector:insert 1:2 in the voltage range of 1.5 to 2.5; ratio of vector:insert 1:3 in the voltage range of 1.0 to 2.5; ratio of vector:insert 1:4 in the voltage range of 0.5 to 2.5; ratio of vector:insert 1:5 in the voltage range of 1.0 to 2.5; and a ratio vector:insert 1:10 in the voltage range of 1.0 to 2.5, with no colonies at 1.5 kV. For the ratio of vector:insert 1:1 the maximum number of columns is th was achieved using a voltage of 2.0 kV. For all other ratios of vector:insert 2.5 kV voltage was optimal. Optimum conditions, apparently, was the ratio of vector:insert 1:10 and a voltage of 2.5 kV. The results of this experiment show that the optimal voltage is 2-2,5 kV, with increased quantities of the insert relative to the vector that do not provide significant advantages over the ratio of 1:2.

Experiment #3:Increased load DNA and high voltage, but not the ratio of vector:insert are critical for maximum efficiency of transformation.

The effect of the amount of DNA on the efficiency of transformation was investigated by electroporation 400 µl of a suspension of yeast, 1, 2, 3, 4, or 8 μg DNA vector while maintaining the same ratio of vector: insert 1:3.S. cerevisiaewere processed as described in example 7. 400 μl of the suspension of yeast cells (1,6 × 109cells/ml) were subjected to electroporation with 1, 2, 3, or 4 μg of the vector in a cell (while maintaining the ratio of vector: insert = 1:3). Referring to figure 4, after 72 hours the number of colonies was determined and represented as (A) transformation efficiency (average number of transformants on ug DNA vector ± standard deviation) and (B) output transformation (the total number of transformants in a cell ± standard deviation). In addition, (a) 400 ál of yeast suspension was subjected to electroporation at 2 or 2.5 kV and different ratios of DNA vector and insert. Transformation efficiency was expressed as the average number of transformants on ug DNA vector.

Interestingly, the transformation efficiency was not significantly changed when up to 4 µg DNA vector used for each transformation, and only slightly decreased when the load 8 µg DNA vector (Figure 4A). As expected, the output size of the transformed library accordingly increased with increasing number of load DNA (Figure 4B). In General, using 4 μg of linearized vector was the most appropriate and effective condition. For the study, is it possible to reduce 12 µg DNA inserts (to achieve a ratio of vector:insert 1:3), tested different ratios of vector:insert. The results show that the ratio can be as low as 1:1.5 to without negative impact on the efficiency of transformation (Figure 4C). However, the observed lower transformation efficiency when the ratio of vector:insert was less than 1:1 or more than 1:5 (data not shown). In addition, it was found that setting the voltage electroporation is critical, because lower voltage electroporation from 2.5 kV to 2 kV or below resulted in a significant loss of efficiency of transformation (Figure 4C and data not presented).

Example 9: the Diversity is the diversity and productivity of libraries of antibodies naive spleen person

The method of electroporation according to the invention is used to obtain a large library of human antibodies. The cDNA fragments VH and VK person from commercially available poly-A RNA spleen separately amplified using polymerase chain reactions (PCR) and previously published primers (Sblattero, D. and Bradbury, A. (1998) Immunotechnology 3:271-8). The Vκ fragments proportionally combined together so that each embryo collection was equally represented in the library. The ScFv fragments separately received from fragments of VH and United Vκ fragments by overlapping PCR. Many yeast libraries were obtained from these scFv fragments, mixed with a linear DNA vector by electroporation and proportionally combined together to maintain an equivalent representation of all germline VH sequences. A large number of yeast colonies sequenced for analysis of germline scFv, and the results confirmed the high diversity. Except in rare germline genes VH7, all other family germline genes were identified, and their performance was roughly proportional to the number of their embryonic genes in the library of the authors of the present invention (see table 8). Because it does not identify identical sequences of VH, appreciated that various the peculiarity library has a 95% chance to be so large, as 1012taking the maximum theoretical diversity 104for Vκ and diversity 108for VH (approximately 108B-cells of the donor), and is limited only by the size of the library. Using this highly effective method of electroporation of yeast is currently possible to construct yeast libraries with sizes approaching typical rahovym libraries (Hoogenboom, H. R. (2002) Methods Mol. Biol. 178:1-37; Sblattero, D. and Bradbury, A. (2000) Nat. Biotechnol. 18:75-80). Indeed, it takes only one day getting a yeast library of 2 × 1010simply scaling electroporation to twenty cuvette, and the yeast library 109currently, it is possible in a conventional manner to obtain for affinity maturation or other optimization purposes.

Table 8
The diversity of the library of antibodies naive spleen person
Embryo collection VHTheoretical distribution (%)The observed diversity of the library of naive spleen person (%) (n=347)
VH12115
VH27 5
VH35062
VH41616
VH4(DP63)2,53,2
VH50,50,3
VH62,51,5
VH70,50

To show that the constructed libraries produce antibodies with reasonably good affinity, were selected libraries of antibodies in the spleen of a person against tumor necrosis factor α of the person (TNFα), first by sorting activated by the magnetic field of the cells to reduce the size of the library to approximately 107cells, then through many cycles FACS, basically as described previously (Chao, G. et al. (2006) Nat. Protoc. 1:755-68; Feldhaus, M. J. et al. (2003) Nat. Biotechnol. 21:163-70). Identified multiple binding TNFα members, and their VH originate from embryonic sequences VH1, VH3 and VH4 man, and of Vκ germline sequences Vκ1 and Vκ2 person that indicates that the library is indeed produces binding antigenre from a diverse pool of antibodies. For one of scFv showed affinity to TNFα 4 nm, and the other showed the greatest affinity to TNFα 40 nm. When turning in IgG binding with 4 nm member kept a similar binding affinity of (1.5 nm in an ELISA), while for linking with 40 nm of the member showed a significant increase in the affinity (0.3 nm by ELISA). This increase in the apparent affinity may be due to avidity turned IgG interacting with the two TNFα in the same homotrimers TNFα protein. Additional selections libraries also identified antibodies with low nanomolar affinity against IL-18 person from this library (data not shown).

Using the most optimal conditions for electroporation can in a conventional manner to achieve the transformation efficiency of yeast 1 × 108the yeast transformants/µg DNA vector. Because of this efficiency transformation reach the minimum amount of cells (100 μl), it is well suited for automation and advance devices for electroporation. For example, you can provide that by adjustment of electroporation in 96-hole tablet for electroporation can reach the size of the library of 9.6 × 109. By repeating this advance electroporation 10-11 times, either manually or automatically, you can easily construct a yeast library of more than 1011transformed for one night. This efficiency and the potential size of the constructed library is approaching the efficiency and size of a typical ragovoy library, and is very desirable in order to maximize success rate using engineered yeast libraries for various applications that can be attached to the library, such as yeast display of antibodies and yeast two-hybrid screening interactions.

References

The incorporation by reference

The contents of all cited references (including literature, patents, patent applications and Internet sites), which may be cited throughout this application, therefore, are expressly listed as references. In the practical implementation of the invention can be applied, unless otherwise indicated, conventional methods of electroporation of cells and biology of the yeast cells are well known in this field.

Equivalents

The invention can be implemented in other specific forms without deviating from its content or essential characteristics. The above options for implementation, thus, should be considered in all respects as illustrative, and not limiting the invention described herein. Volume is subramania, thus, specified in the attached claims and not the foregoing description, and all changes within the meaning and range of equivalency of the claims therefore are intended to include.

1. The method of obtaining a yeast library, where the method comprises the stages:
incubation of yeast cells in a solution containing from 0.01 to 1.0 M lithium acetate (LiAc) and from 1 to 100 mm dithiothreitol (DTT);
obtaining a suspension containing the linearized DNA vector and DNA insert in the ratio of from 1:0.5 to 1:10, sorbitol and CaCl2or MgCl2and insert DNA contains sufficient 5' and 3' vector sequence for the effective conduct of homologous recombination, and where to use the 4 µg DNA vector 400 ál of 1.6×109yeast cells/ml, and
electroporation solution of yeast cells suspension containing linearized DNA vector and DNA insert at a voltage of 0.5 kV/cm to 12.5 kV/cm with a capacity from 10 to 50 UF.

2. The method according to claim 1, where the suspension containing 1 M sorbitol and 1 mm CaCl2.

3. The method according to claim 1, where the suspension containing 1 M sorbitol and 1 mm MgCl2.

4. The method according to claim 1, where the yeast cells are incubated in a solution containing cold 0.1 M LiAc, and 10 mm DTT.

5. The method according to claim 1, where the suspension is subjected to electroporation at 2.5 kV/cm with a capacity of 25 μf.

6. The method according to claim 1, where the stage elec is reparatii carried out in a ditch with a clearance of 0.2 see

7. The method according to claim 1, where the suspension contains 400 µl of 1.6×109yeast cells/ml

8. The method according to claim 1, where the ratio of the DNA vector to DNA insert is 1:3.

9. The method of obtaining a yeast library, where the method comprises the stages:
cultivation of yeast cells to OD600from 1.0 to 2;
washing of yeast cells in water;
washing the yeast cells in a solution containing sorbitol and CaCl2;
incubation of yeast cells in a second solution containing from 0.01 to 1.0 M lithium acetate (LiAc) and from 1 to 100 mm dithiothreitol (DTT);
washing the yeast cells in a third solution containing sorbitol and CaCl2;
resuspendable of yeast cells in a solution containing sorbitol and CaCl2or MgCl2for the formation of a suspension of yeast cells for electroporation;
add a suspension of yeast cells for electroporation of suspension containing linearized DNA vector and DNA insert in the ratio of from 1:0.5 to 1:10, sorbitol and CaCl2or MgCl2and insert DNA contains sufficient 5' and 3' vector sequence for the effective conduct of homologous recombination, and where to use the 4 µg DNA vector 400 ál of 1.6×109yeast cells/ml to form a suspension of yeast cells - DNA for electroporation, and
electroporation of suspension of yeast cells - DNA for electroporation suspension containing linearized the first DNA vector and DNA insert, at a voltage of 2.5 kV/cm to 12.5 kV/cm in a ditch with a clearance of 0.2 see

10. The method according to claim 9, where the fourth solution containing 1 M sorbitol and 1 mm CaCl2.

11. The method according to claim 9, where the fourth solution containing 1 M sorbitol and 1 mm MgCl2.

12. The method according to claim 9, where the second solution contains cold 0.1 M LiAc, and 10 mm DTT.

13. The method according to claim 9, where the suspension of yeast cells - DNA for electroporation, subjected to electroporation at 2.5 kV/cm with a capacity of 25 μf.

14. The method according to claim 9, where the suspension contains 400 µl of 1.6×109yeast cells/ml

15. The method according to claim 9, where the ratio of the DNA vector to DNA insert is 1:3.



 

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