Scalable fermentation process

Abstract

This invention provides a robust fermentation process for the expression of a capsid protein of a bacteriophage which is forming a VLP by self-assembly, wherein the process is scalable to a commercial production scale and wherein the expression rate of the capsid protein is controlled to obtain improved yield of soluble capsid protein. This is achieved by combining the advantages of fed-batch culture and of lactose induced expression systems with specific process parameters providing improved repression of the promoter during the growth phase and high plasmid retention throughout the process.

1 . A process for expression of a recombinant capsid protein of a RNA bacteriophage being capable of forming a virus-like particle (VLP) by self-assembly, said process comprising the steps of:
 a.) introducing an expression plasmid into a bacterial host, wherein said expression plasmid comprises an expression construct, wherein said expression construct comprises (i) a first nucleotide sequence encoding said recombinant capsid protein, or mutant or fragment thereof, and (ii) a promoter being inducible by lactose;   b.) cultivating said bacterial host in a medium comprising a major carbon source;   wherein said cultivating is performed in batch culture and under conditions under which said promoter is repressed by lacI, wherein said lacI is overexpressed by said bacterial host;   c.) feeding said batch culture with said major carbon source; and   d.) inducing said promoter with an inducer, wherein said feeding of said batch culture with said major carbon source is continued.   
     
     
         2 . (canceled) 
     
     
         3 . The process of  claim 1 , wherein said bacteriophage is selected from the group consisting of:
 a.) bacteriophage Qβ;   b.) bacteriophage AP205;   c.) bacteriophage fr;   d.) bacteriophage GA;   e.) bacteriophage SP;   f.) bacteriophage MS2;   g.) bacteriophage M11;   h.) bacteriophage MX1;   i.) bacteriophage NL95;   j.) bacteriophage f2;   k.) bacteriophage PP7 and   l.) bacteriophage R17.   
     
     
         4 . The process of  claim 1 , wherein said RNA bacteriophage is Qƒ3. 
     
     
         5 . The process of  claim 1 , wherein said recombinant capsid protein has the amino acid sequence of SEQ ID NO:5. 
     
     
         6 . The process of  claim 1 , wherein said expression construct comprises a first stop codon and a second stop codon, wherein said first stop codon is located directly 3′ of said first nucleotide sequence and wherein said second stop codon is located directly 3′ of said first stop codon, and wherein at least one of said first or second stop codon is TAA. 
     
     
         7 . The process of  claim 1 , wherein said expression construct comprises a first nucleotide sequence and a second nucleotide sequence, wherein said first nucleotide sequence is encoding Qβ coat protein (CP), and wherein said second nucleotide sequence is encoding the Qβ A1 protein or a mutant or fragment thereof, and wherein said first and said second nucleotide sequence are separated by exactly one sequence stretch comprising at least one TAA stop codon. 
     
     
         8 . The process of  claim 1 , wherein said expression construct comprises or alternatively consists of the nucleotide sequence of SEQ ID NO:6. 
     
     
         9 . The process of  claim 1 , wherein said expression plasmid comprises the nucleotide sequence of SEQ ID NO:1. 
     
     
         10 . (canceled) 
     
     
         11 . The process of  claim 1 , wherein said promoter is selected from the group consisting of the
 a.) tac promoter;   b.) trc promoter;   c.) tic promoter;   d.) lac promoter;   e.) lacUV5 promoter;   f.) P syn  promoter;   g.) lpp a  promoter;   h.) lpp-lac promoter;   i.) T7-lac promoter;   i.) T3-lac promoter;   k.) T5-lac promoter; and   l.) a promoter having at least 50% sequence homology to SEQ ID NO:2.   
     
     
         12 . The process of  claim 1 , wherein said promoter comprises the nucleotide sequence of SEQ ID NO:2. 
     
     
         13 . The process of  claim 1 , wherein said major carbon source is glycerol. 
     
     
         14 . The process of  claim 1 , wherein said feeding of said batch culture is performed with a flow rate, wherein said flow rate increases with an exponential coefficient μ. 
     
     
         15 . The process of  claim 1 , wherein said inducing of said promoter is performed by co-feeding said batch culture with said inducer and said major carbon source at a constant flow rate. 
     
     
         16 . The process of  claim 1 , wherein said inducing of said promoter is performed by co-feeding said batch culture with said inducer and said major carbon source at an increasing flow rate. 
     
     
         17 . The process of  claim 15 , wherein said inducer is lactose and wherein said lactose and said major carbon source are co-fed to said batch culture in a ratio of about 2:1 to 1:4 (w/w). 
     
     
         18 . The process of  claim 1 , wherein said inducer is IPTG and wherein the concentration of said IPTG in said medium is 0.001 to 5 mM. 
     
     
         19 . (canceled) 
     
     
         20 . The process of  claim 1 , wherein said lacI is overexpressed by said bacterial host, wherein said overexpression is caused by lacI q  or lacQ1. 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . The process of  claim 1 , wherein said inducer is lactose and wherein said bacterial host comprises β-galactosidase activity. 
     
     
         24 . The process of  claim 1 , wherein said cultivating and said feeding of said batch culture and said inducing of said promoter is performed at a temperature which is below the optimal growth temperature of said bacterial host. 
     
     
         25 . The process of  claim 1  wherein:
 a.) said major carbon source is glycerol; 
 b.) said feeding of said batch culture is performed with a flow rate, wherein said flow rate increases with an exponential coefficient μ, and wherein said exponential coefficient μ is below μ max ; 
 c.) said inducer is lactose; 
 d.) and said lactose and said major carbon source are co-fed to said batch culture in a ratio of 2:1 to 1:4 (w/w); 
 e.) said bacterial host is  E. coli  RB791; and 
 f.) said cultivating and feeding of said batch culture and said inducing of said promoter is performed at a temperature of about 30° C. 
 
     
     
         26 . (canceled) 
     
     
         27 . The process of  claim 1 , wherein throughout steps b.) to d.) of said process oxygen is supplied to said bacterial host, wherein said oxygen supply is effected such that the partial pressure of oxygen in the medium (pO 2 ) is at least about 40%.