Method for producing soluble recombinant interferon protein without denaturing
AssigneePfenex Inc.
InventorsJeffrey Allen, Ping-Hua Feng, Anant Patkar, Keith L. Haney, Lawrence Chew, Lei Lei Phokham Sengchanthalangsy
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Abstract
The present invention relates to the field of recombinant protein production in bacterial hosts. It further relates to extraction of soluble, active recombinant protein from an insoluble fraction without the use of denaturation and without the need for a refolding step. In particular, the present invention relates to a production process for obtaining high levels a soluble recombinant Type 1 interferon protein from a bacterial host.
Claims28
A method for producing a recombinant Type 1 interferon protein, said method comprising:
- expressing the recombinant interferon protein by culturing a Pseudomonas or E. coli host cell containing an expression construct comprising a coding sequence that has been optimized for expression in the host cell;
- lysing the host cell;
- obtaining an insoluble fraction and a soluble fraction from the lysis step;
- extracting the insoluble fraction by subjecting it to non-denaturing extraction conditions;
- and
- obtaining an extract pellet and an extract supernatant from the insoluble fraction;
- wherein the recombinant protein in the extract supernatant is present in soluble form, active form, or a combination thereof, without being further subjected to a renaturing or refolding step.
A method for extracting a recombinant Type 1 interferon protein, wherein the recombinant interferon protein is present in an insoluble fraction, said insoluble fraction produced after lysis of a Pseudomonas or E. coli host cell expressing the recombinant interferon protein, said method comprising:
- subjecting the insoluble fraction to non-denaturing extraction conditions;
- and
- obtaining an extract pellet from the insoluble fraction, said extract pellet comprising recombinant interferon protein;
- wherein the recombinant interferon protein in the extract pellet is in soluble form, active form, or a combination thereof, without being subjected to a renaturing or refolding step.
A method for producing an insoluble fraction comprising a recombinant Type 1 interferon protein, wherein the recombinant interferon protein is expressed in a Pseudomonas or E. coli host cell from a nucleic acid construct comprising a nucleic acid sequence that is operably linked to a lac derivative promoter, said method comprising:
- growing the host cell at a temperature of about 25° C. to about 33° C. and at a pH of about 5.7 to about 6.5, to an OD600 of about 80 to about 160;
- and
- inducing the host cell at a concentration of about 0.08 mM to about 0.4 mM IPTG;
- lysing the host cell and centrifuging it to produce the pellet fraction;
- wherein soluble, active, or soluble and active recombinant interferon protein can be obtained by extracting the pellet fraction under non-denaturing conditions without a subsequent renaturing or refolding step.
Dependencies are inferred from the claim text.