Protein expression strains

Abstract

The invention provides an improved host strain for production of desired protein.

1 . A fungal host cell having:
 a. a modified Not4 protein or homolog thereof, or   b. a modified activity level of Not4 protein or homolog thereof, or   c. a modified NOT4 gene or homolog thereof, or   d. a modified level of expression of NOT4 gene, or homolog thereof.   
     
     
         2 . The fungal host cell of  claim 1 , wherein the modified level is a reduced level. 
     
     
         3 . The fungal host cell of  claim 2 , wherein the reduced level is relative to the level to a reference fungal host cell, such as a fungal host cell in which the Not4 protein comprises or consists of SEQ ID NO: 2. 
     
     
         4 . The fungal host cell according to  claim 1 , which is a yeast or a filamentous fungus. 
     
     
         5 . The fungal host cell according to  claim 1 , comprising a nucleotide sequence encoding a desired protein such as heterologous protein, such as a serum protein, preferably an albumin or variant, fragment and/or fusion thereof. 
     
     
         6 . The fungal host cell according to  claim 1 , in which the Not4 protein comprises a mutation at position corresponding to a position selected from 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469 or 470 of SEQ ID NO: 2, preferably a position selected from 429, 430, 434, or 437. 
     
     
         7 . The fungal host cell according to  claim 6  in which the mutation at a position corresponding to position 429 of SEQ ID NO: 2 is a substitution to A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y, preferably to G, A, V, L or I, more preferably to I, L or V, most preferably to I. 
     
     
         8 . The fungal host cell according to  claim 6  in which the mutation at a position corresponding to position 429 of SEQ ID NO: 2 is F429I. 
     
     
         9 . The fungal host cell according to  claim 1 , comprising a polynucleotide encoding SEQ ID NO: 4. 
     
     
         10 . The fungal host cell according to  claim 1 , which lacks a NOT4 gene, or homolog thereof, or Not4 protein, or homolog thereof. 
     
     
         11 . The fungal host cell according to  claim 1 , in which the host cell is a  Saccharomyces  such as  Saccharomyces cerevisiae.    
     
     
         12 . A method for producing a desired protein, such as a heterologous protein, from a fungal host cell, said method comprising:
 (i) providing a fungal host cell according to  claim 1 ,   (ii) culturing the fungal host cell or a culture thereof to produce the desired protein,   (iii) optionally recovering the desired protein,   (iv) optionally purifying the desired protein,   (v) optionally formulating the desired protein with a therapeutically acceptable carrier or diluent thereby to produce a therapeutic product suitable for administration to a human or an animal, and   (vi) optionally providing the desired protein in unit dosage form.   
     
     
         13 . A method for increasing the yield of a desired protein (such as a heterologous protein) comprising:
 (i) providing a fungal host cell (such as a yeast or a filamentous fungus) having:
 a. a modified Not4 protein or homolog thereof, or 
 b. a modified level of activity (preferably reduced) of Not4 protein or homolog thereof, or 
 c. a modified NOT4 gene or homolog thereof, or 
 d. a modified level of expression (preferably reduced) of NOT4 gene, or homolog thereof, 
   (ii) culturing the host cell to produce the desired protein, and   (iii) optionally recovering the desired protein,   (iv) optionally purifying the desired protein,   (v) optionally formulating the desired protein with a therapeutically acceptable carrier or diluent thereby to produce a therapeutic product suitable for administration to a human or an animal, and   (vi) optionally providing the desired protein in unit dosage form.   
     
     
         14 . The method according to  claim 12 , in which the yield of the desired protein is at least 2% higher than the yield from a reference fungal host cell such as a fungal host cell having Not4 protein of SEQ ID NO: 2. 
     
     
         15 . The method according to  claim 12 , in which the desired protein is an albumin or variant, fragment and/or fusion thereof. 
     
     
         16 . The method according to  claim 12 , in which the host cell is cultured at a scale of at least 5 L. 
     
     
         17 . (canceled)