Method for producing recombinant proteins using recombinant brevibacillus
The present invention provides a method for producing a recombinant protein, which can increase the cell growth rate in culture while stably maintaining plasmids in the cells, thereby improving the recombinant protein yield. The present invention provides a method for producing a recombinant protein, including a high-temperature culture step of culturing at 32° C. or higher a Brevibacillus bacterium that contains a gene encoding a recombinant protein, and a low-temperature culture step of culturing the Brevibacillus bacterium at a temperature lower than 32° C. after the high-temperature culture step.
1. A method for producing a recombinant protein, comprising:
a high-temperature culture step of culturing in a liquid medium at 34° C. or higher and 40° C. or lower a Brevibacillus choshinensis bacterium that contains a gene encoding the recombinant protein;
a temperature shifting step of reducing the temperature of the liquid medium prepared in the high-temperature culture step without replacing the liquid medium, and
a low-temperature culture step of culturing the Brevibacillus choshinensis bacterium in the liquid medium at a temperature at 25° C. or higher and lower than 32° C., which is lower than the culturing temperature in the high-temperature culture step by at least 2° C.-8° C.
2. The method for producing a recombinant protein according to claim 1 ,
wherein a shift from the high-temperature culture step to the low-temperature culture step is made between the early and mid-exponential growth phases of the Brevibacillus bacterium.
3. The method for producing a recombinant protein according to claim 1 ,
wherein the recombinant protein is an antibody-binding protein.
4. The method for producing a recombinant protein according to claim 3 ,
wherein the antibody-binding protein is selected from the E domain, D domain, A domain, B domain, or C domain of Protein A, the C domain or D domain of Protein Cy the B domain of Protein L, proteins comprising the foregoing domains connected together, or functional variants of the foregoing.
5. The method for producing a recombinant protein according to claim, 1 wherein the recombinant protein is a bioactive protein.
6. The method for producing a recombinant protein according to claim 5 ,
wherein the bioactive protein is a peptide hormone or a precursor thereof.
7. The method for producing a recombinant protein according to claim 1 ,
wherein the recombinant protein is an antibody or antibody-like molecule.
8. The method for producing a recombinant protein according to claim 2 , wherein the recombinant protein is an antibody-binding protein.
9. The method for producing a recombinant protein according to claim 8 ,
wherein the antibody-binding protein is selected from the E domain, D domain, A domain, B domain, or C domain of Protein A, the C domain or D domain of Protein Cy the B domain of Protein L, proteins comprising the foregoing domains connected together, or functional variants of the foregoing.
10. The method for producing a recombinant protein according to claim 2 , wherein the recombinant protein is a bioactive protein.
11. The method for producing a recombinant protein according to claim 10 ,
wherein the bioactive protein is a peptide hormone or a precursor thereof.
12. The method for producing a recombinant protein according to claim 2 , wherein the recombinant protein is an antibody or antibody-like molecule.