KEX2 cleavage regions of recombinant fusion proteins

Abstract

The invention relates to a fusion DNA construct comprising a KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5′ to the KEX2 site, a fusion polypeptide, vectors and cells comprising the fusion DNA construct, methods for producing desired proteins from filamentous fungal cells and methods for enhancing the secretion and/or cleavage of a desired protein from a cell.

1 . A fusion DNA construct encoding a fusion polypeptide comprising in operable linkage from the 5′ end of said construct,
 a. promoter,   a first DNA molecule encoding a signal sequence,   a second DNA molecule encoding a carrier protein,   a third DNA molecule encoding a KEX2 region, said region comprising a KEX2 site (B 1 B 2 ) and a KEX2 site pre-sequence ((X)n=2 to 6) immediately 5′ to the KEX2 site; and   a fourth DNA molecule encoding a desired protein.   
   
   
       2 . The fusion DNA construct of  claim 1 , wherein the KEX2 region is X 4 X 3 X 2 X 1 KR. 
   
   
       3 . The fusion DNA construct of  claim 2 , wherein
 X 4  is V, S, N, L, or K;   X 3  is A, V, D, W, E or P;   X 2  is V, I, L or F; and   X 1  is E, S, T or Y.   
   
   
       4 . The fusion DNA construct of  claim 3 , wherein X 4  is V. 
   
   
       5 . The fusion DNA construct of  claim 3 , wherein X 3  is A. 
   
   
       6 . The fusion DNA construct of  claim 3 , wherein X 2  is V. 
   
   
       7 . The fusion DNA construct of  claim 3 , wherein X 1  is E or Y. 
   
   
       8 . The fusion DNA construct of  claim 3 , wherein the KEX2 site pre-sequence is selected from the group consisting of VAVE (SEQ ID NO: 84); VAVY (SEQ ID NO: 87); LAVE (SEQ ID NO: 88); KAVE (SEQ ID NO: 89); VAIE (SEQ ID NO: 90); VALE (SEQ ID NO: 91); VAFE (SEQ ID NO: 92); VWVE (SEQ ID NO: 93); VEVE (SEQ ID NO: 94); and VPVE (SEQ ID NO: 95). 
   
   
       9 . The fusion-DNA construct of  claim 1 , wherein the first DNA molecule and second DNA molecule encode a  Trichoderma  CBH1 signal sequence and carrier protein or a  Trichoderma  endoglucanase signal sequence and carrier protein. 
   
   
       10 . The fusion DNA construct of  claim 1 , wherein the first DNA molecule and second DNA molecule encode a glucoamylase signal sequence and carrier protein or an alpha amylase signal sequence and carrier protein. 
   
   
       11 . The fusion DNA construct of  claim 1 , wherein the desired protein is an enzyme. 
   
   
       12 . The fusion DNA construct of  claim 1 , wherein the desired protein is a therapeutic protein. 
   
   
       13 . The fusion DNA construct of  claim 12 , wherein the therapeutic protein is an antibody. 
   
   
       14 . The fusion DNA construct of  claim 13 , wherein the antibody is a light chain or heavy chain monoclonal antibody. 
   
   
       15 . The fusion DNA construct of  claim 3 , wherein the first DNA molecule and second DNA molecule encode a CBH1 signal sequence and carrier protein and the fourth DNA molecule encodes an antibody light chain or fragment thereof. 
   
   
       16 . The fusion DNA construct of  claim 3 , wherein the first DNA molecule and second DNA molecule encode a glucoamylase signal sequence and carrier protein and the fourth DNA molecule encodes an antibody light chain or fragment thereof. 
   
   
       17 . The fusion protein encoded by the fusion DNA construct of  claim 1 . 
   
   
       18 . A host cell comprising the fusion DNA construct of  claim 1 . 
   
   
       19 . The host cell of  claim 18 , wherein said host cell is a  Trichoderma  host cell. 
   
   
       20 . The host cell of  claim 19 , wherein the  Trichoderma  cell is a  T. reesei  cell. 
   
   
       21 . A vector comprising the fusion DNA construct of  claim 1 . 
   
   
       22 . A host cell comprising the vector of  claim 21 . 
   
   
       23 . The host cell of  claim 22 , wherein the host cell is a  Trichoderma  host cell. 
   
   
       24 . A process for producing a desired protein in a filamentous fungal cell comprising:
 a) obtaining a filamentous fungal host cell comprising a fusion DNA construct according to  claim 1 ;   b) culturing the host cell under suitable conditions which allow for the expression and production of the desired protein; and   c) recovering the desired protein.   
   
   
       25 . The process according to  claim 24 , wherein the host cell is a  Trichoderma  strain. 
   
   
       26 . The process according to  claim 25 , wherein the  Trichoderma  cell is a  T. reesei  host cell. 
   
   
       27 . The process according to  claim 24 , wherein the desired protein is an immunoglobulin. 
   
   
       28 . The process according to  claim 27 , wherein the immunoglobulin is a monoclonal antibody. 
   
   
       29 . The process according to  claim 28 , wherein the monoclonal antibody is a light chain or heavy chain monoclonal antibody or fragment thereof. 
   
   
       30 . The process according to  claim 28 , wherein the host cell is a  Trichoderma  cell, the desired protein is a light chain antibody and the KEX2 region is X 4 X 3 X 2 X 1 KR and X 4  is V and X 1  is E, S, T or Y. 
   
   
       31 . A method for cleaving a desired protein from a recombinant fusion polypeptide comprising expressing in a filamentous fungal cell a fusion polypeptide encoded by a fusion DNA construct according to  claim 1 , wherein said KEX2 region provides a protein cleavage site and obtaining a desired protein which is cleaved from the expressed fusion polypeptide. 
   
   
       32 . The method according to  claim 31 , wherein the desired protein is a therapeutic protein. 
   
   
       33 . The method according to  claim 31 , wherein the desired protein is an antibody. 
   
   
       34 . The method according to  claim 31 , wherein the cleavage of the desired protein is increased compared to the cleavage of said desired protein from an equivalent fusion polypeptide lacking the KEX2 site pre-sequence. 
   
   
       35 . The method according to  claim 26 , wherein the KEX2 region is X 4 X 3 X 2 X 1 KR and X 4  is V and X 1  is E, S, T or Y. 
   
   
       36 . A method for increasing the production of an antibody from a filamentous fungal cell comprising obtaining a filamentous fungal cell comprising a fusion DNA construct of  claim 3 , culturing the fungal cell under suitable conditions for expression of the fusion polypeptide and allowing secretion of the fusion polypeptide, wherein the secretion of the desired protein is increased compared to the secretion of an equivalent fusion polypeptide not including a KEX2 pre-sequence. 
   
   
       37 . The method of  claim 36 , wherein the secretion of the desired protein is increased by at least 30% compared to the secretion of the desired protein from the equivalent fusion polypeptide. 
   
   
       38 . A fusion polypeptide comprising from an amino terminus of said fusion polypeptide a first amino acid sequence comprising:
 a) a signal sequence functional as a secretory sequence;   b) a second amino acid sequence comprising a carrier protein;   c) a third amino acid sequence comprising a KEX2 region, said region comprising a KEX2 site (B 1 B 2 ) and a KEX2 site pre-sequence ((X)n=2 to 6) immediately 5′ to the KEX2 site; and   d) a fourth amino acid sequence comprising a desired protein. wherein B 1  or B 2  is K or R and X is any amino acid residue.   
   
   
       39 . A method for identifying enhanced secretion and/or cleavage of a desired protein comprising:
 a) altering a KEX2 site pre-sequence of a parental fusion polypeptide, said parental fusion polypeptide comprising a signal sequence; a KEX2 region comprising a KEX2 site (B 1 B 2 ) and a KEX2 site pre-sequence ((X)n=4 which is located immediately N-terminal to said KEX2 site, and an amino acid sequence comprising a desired protein to produce a set of test recombinant fusion polypeptides that are identical to said parental fusion polypeptide except for said KEX2 site pre-sequence;   b) evaluating secretion and/or cleavage of said test fusion polypeptides and said parental fusion polypeptide by a filamentous fungal cell; and   c) identifying a test fusion polypeptide that has enhanced secretion and/or cleavage as compared to said parental fusion polypeptide.   
   
   
       40 . The method according to  claim 39  further comprising identifying an optimized KEX2 site pre-sequence which comprises, testing a plurality of different test fusion polypeptides, and determining which of said different test fusion polypeptides has greater secretion and/or protein cleavage, wherein said optimized KEX2 site pre-sequence is the altered KEX2 site pre-sequence of the test recombinant fusion polypeptide that has the greatest secretion and/or protein cleavage.