METHOD FOR PREPARING ELECTROCOMPETENT YEAST CELLS, AND METHOD FOR USING SAID CELLS

Abstract

The present invention relates to improved yeast transformation of yeast cells and yeast cell libraries transformed thereby. More specifically, the present invention relates to the transformation of yeast by electroporation.

Claims

1. A yeast surface display library comprising electrocompetent yeast cells comprising a transformation efficiency of higher than about 2×10.sup.8 transformants/μg DNA/100 μl cell volume, wherein said electrocompetent yeast cells are produced by a method comprising a) growing yeast cells to an OD.sub.600 of between 1.0 to 2; b) washing the cells with cold water; c) washing the cells with a cold solution comprising sorbitol and CaCl.sub.2; d) incubating the cells in a solution comprising lithium acetate and tris2-carboxyethyl)phosphine (TCEP); e) washing the cells with a cold solution comprising sorbitol and CaCl.sub.2; f) resuspending the cells in a solution comprising sorbitol; g) washing the cells with a cold solution comprising sorbitol; h) mixing the cells with the DNA to be transfected, to form a pre-electroporation-mix; i) transferring said pre-electroporation-mix into a suitable electroporation cuvette; and j) electroporating said cells at between 2.5 kV/cm to 12.5 kV/cm for between 2 to 5 MS.

2. The yeast surface display library of claim 1, wherein said DNA is linear or circular.

3. The yeast surface display library of claim 1, wherein said DNA comprises a library of DNA fragments encoding for a library of proteins of interest.

4. The yeast surface display library of claim 3, wherein said library of proteins of interest is a T-cell receptor (TCR) library.

5. The yeast surface display library of claim 4, wherein the diversity of said library is higher than 10.sup.12.

6. The yeast surface display library of claim 4, wherein the TCR library comprises a single chain TCR (scTCR).

7. The yeast surface display library of claim 6, wherein the scTCR is a Vβ-linker-Vα scTCR.

8. The yeast surface display library of claim 6, wherein the scTCR is a Vα-linker-Vβ scTCR.

9. The yeast surface display library of claim 6, wherein the scTCR is fused with a self-cleaving peptide.

10. The yeast surface display library of claim 9, wherein the self-cleaving peptide comprises a 2A-peptide.

11. The yeast surface display library of claim 1, wherein the concentration of sorbitol is about 0.1 to about 10 M.

12. The yeast surface display library of claim 1, wherein the concentration of CaCl.sub.2 is about 0.1 to about 10 mM.

13. The yeast surface display library of claim 1, wherein the concentration of lithium acetate is about 0.01 to about 1.0 M.

14. The yeast surface display library of claim 1, wherein the concentration of TCEP is about 1 to about 100 mM.

15. The yeast surface display library of claim 1, wherein the yeast cell is of Saccharomyces genus.

16. The yeast surface display library of claim 1, wherein the yeast cell is of Schizosaccharomyces genus.

17. The yeast surface display library of claim 1, wherein the method further comprises k) diluting the electroporated cells into a 1:1 mix of a solution of sorbitol in growth medium; l) resuspending the cells in a suitable growth medium; m) optionally, performing dilutions for a calculation of diversity, and plating said dilutions on SD-CAA plates containing kanamycin; n) expanding said library per electroporation in the suitable growth medium; and o) optionally, suitably storing said expanded library.

18. A method of identifying an antigen binding molecule with increased antigen binding affinity, comprising, a) providing a yeast surface display library comprising electrocompetent yeast cells, wherein said electrocompetent yeast cells are produced by a method comprising a1) growing yeast cells to an OD.sub.600 of between 1.0 to 2; a2) washing the cells with cold water; a3) washing the cells with a cold solution comprising sorbitol and CaCl.sub.2; a4) incubating the cells in a solution comprising lithium acetate and tris2-carboxyethyl)phosphine (TCEP); a5) washing the cells with a cold solution comprising sorbitol and CaCl.sub.2; a6) resuspending the cells in a solution comprising sorbitol; a7) washing the cells with a cold solution comprising sorbitol; a8) mixing the cells with a library of DNA fragments encoding a library of antigen binding molecules, to form a pre-electroporation-mix; a9) transferring said pre-electroporation-mix into a suitable electroporation cuvette; and a10) electroporating said cells at between 2.5 kV/cm to 12.5 kV/cm for between 2 to 5 ms, and b) analyzing antigen binding affinity of the yeast surface display library, and c) identifying the antigen binding molecule with increased antigen binding affinity.

19. A kit for preparing electrocompetent yeast cells, comprising yeast cells, a first solution comprising sorbitol and CaCl.sub.2 or MgCl.sub.2, a second solution comprising lithium acetate (LiAc) and tris2-carboxyethyl)phosphine (TCEP), and instructions for preparing said electrocompetent yeast cells using said first and second solutions.

20. The kit of claim 19, wherein said instructions comprise: a) growing the yeast cells to an OD.sub.600 of between 1.0 to 2; b) washing the cells with cold water; c) washing the cells with a cold solution comprising the sorbitol and CaCl.sub.2; d) incubating the cells in a solution comprising the LiAc and the TCEP; e) washing the cells with a cold solution comprising the sorbitol and CaCl.sub.2; f) resuspending the cells in a solution comprising the sorbitol; and g) optionally, suitably storing said cells.

Description

[0056] Other preferred embodiments can be derived from the examples with reference to the FIGURES as described herein, nevertheless, without being limited thereto. For the purposes of the invention, all references as cited herein are incorporated by reference in their entireties.

[0057] FIG. 1 shows a comparison of the improved transfection efficiency of TCEP, when compared with DTT under the same conditions.

EXAMPLES

[0058] The practice of the invention employs, unless otherwise indicated, conventional techniques of cellular electroporation and yeast cell biology, which are well known in the art.

I. Media

1. YPD Media

[0059]

TABLE-US-00001 Yeast extract 10 g Bacto-peptone 20 g Dextrose 20 g bring volume to 1 L with H.sub.2O (ad sterile glucose to autoclaved solution)

2. SD-CAA (pH 4.5):

[0060]

TABLE-US-00002 Sodium citrate dihydrate 14.8 g (50 mM final) Citric acid monohydrate  4.2 g (20 mM final) in 800 mL of H.sub.2O, autoclave. Casamino acids 5.0 g Yeast nitrogen base (without amino acids) 6.7 g Glucose  20 g bring volume to 1 L with H.sub.2O and sterile filter

3. SD-CAA Plates:

[0061]

TABLE-US-00003 Sorbitol 182.2 g Agar 15 g Sodium citrate 14.8 g Citric acid monohydrate 4.2 g in 800 mL of H.sub.2O, autoclave, and cool to ~55° C. Casamino acids 5.0 g Yeast nitrogen base (without amino acids) 6.7 g Glucose 20 g Kanamycin sulfate 35 mg in 200 ml H.sub.2O and sterile filter, add to cooled autoclaved solution

II. Preparation of Electrocompetent Yeast Cells

[0062] 20 μl of freshly thawed yeast stock from −80° C. were streaked out on YPD agar plates, and incubated for two days at 30 C. Single colonies (take whole colony) were taken from the YPD agar plate into 15 ml YPD media, and shaking was performed over night at 30° C. Next morning, 10 ml of culture were transferred into 100 ml fresh YPD medium, and shaking was continued for 7 h at 30 C. The OD.sub.600 was determined and 1 l cold YPD medium was inoculated to an OD.sub.600 of 0.2. The shaker flask was placed in a precooled (4° C.) shaker. The shaker was programmed to start heating (30° C.) and shaking (250 rpm) 5 h before work day begins. Incubation was performed until OD.sub.600 reached 1.5 which was usually 6 h after shaking started.

[0063] Subsequent steps have to be performed on ice and with cooled solutions, tubes, cuvettes and centrifuge, if not stated otherwise.

[0064] The cells were pelleted at 2,000 g and 4° C. for 3 mM (2 step process in 10 Falcon tubes, 50 ml), washed twice with 25 ml cold H.sub.2O and pelleted at 2,000 g for 3 min. The cells were washed with 25 ml of cold sorbitol, 1 M/CaCl.sub.2, 1 mM; and pelleted at 2,000 g for 3 min. The cells were resuspended in 25 ml lithium acetate, 100 mM/TCEP, 10 mM. A 50 ml Falcon tubes with filter lid was used to allow for aeration; the cells were incubated at 30° C. while shaking at 160 rpm for 30 min, placed on ice and the cells were pelleted at 2,000 g and 4° C. for 3 min. The cells were washed with 25 ml of cold 1 M sorbitol/1 mM CaCl.sub.2; and pelleted at 2,000 g and 4° C. for 3 min, and washed with 25 ml of cold 1 M sorbitol; and pelleted at 2,000 g and 4° C. for 3 min. The cells were suspended in a conical tube in cold 1 M sorbitol to a final volume of 400 μl per electroporation reaction. Electrocompetent cells can be stored directly at −80° C. Before using the samples for electroporation, leaked salts have be removed by centrifugation (2,000 g, 4° C., 5 mM) and washing twice with cold sorbitol, 1 M.

III. Electroporation

[0065] 400 μl of cells were mixed with 5-10 μl DNA (vector) in H.sub.2O, kept on ice for 3 min and transferred to a precooled 0.2 cm electroporation cuvette. Using a BioRad MicroPulser Electroporation System, the cells were electroporated at 2.5 kV. Typical time constants were at about 4 ms, preferably at 4 ms. The electroporated cells were transferred into 10 ml of 1:1 mix of 1 M sorbitol:YPD media at 30° C. for 1 hour without shaking. The cells were harvested at 2,000 g for 3 min at room temperature, and resuspended in 10 ml SD-CAA at room temperature. Dilutions were performed for calculation of diversity (1:10.sup.5 to 1:10.sup.7). Dilutions on SD-CAA plates containing kanamycin were incubated for 1 day at 30° C. and for three days at room temperature. The library was transferred into 100 ml SD-CAA (preferably per electroporation) and shaking was continued for 24 h at 30° C. at 160 rpm. Expanded libraries can be used directly for induction or stored at 4° C. for two weeks. Long term storage can be performed by freezing in 30% glycerol at −80° C.

[0066] By using the most optimal electroporation condition, one can routinely achieve yeast transformation efficiency of about 2×10.sup.8 yeast transformants/μg vector DNA (see FIG. 1). As this transformation efficiency is achieved in minimal cell volume (100 μl), it is highly amenable to automation and multiwell electroporation devices.