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.

1 . A yeast surface display library comprising electrocompetent yeast cells comprising a transformation efficiency of higher than about 2×10 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 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 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 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 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 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 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 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 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 2  or MgCl 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 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 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 2 ;   f) resuspending the cells in a solution comprising the sorbitol; and   g) optionally, suitably storing said cells.