Method for obtaining single chain antibodies to human interferon α2b
View Patent ↗A bacterial high-expression system which is applicable for simultaneous screening of large numbers of recombinant clones from combinatorial antibody libraries is disclosed. The method pertains to screening of single chain antibodies from libraries expressed in the periplasm of E. coli by secretion. By this approach, approximately 10 4 clones can be screened in a single round. After screening, the clones, which express the recombinant antibodies to the desired antigen, can be directly used for production of large quantities of antibodies from microorganism culture. The system is especially attractive for fast screening of antibody libraries from a hybridoma source. A refolding method for the large-scale production of biologically active scFv-6 his proteins from bacterial inclusion bodies is also disclosed.
1. A method for recovery of a ScFv antibody having the sequence shown in SEQ ID NO:2 or having the sequences shown in SEQ ID NOS: 3 and 4 from E. coli inclusion bodies in biologically active form which comprises:
providing a transformed E. coli cell expressing a nucleic acid sequence encoding the ScFv antibody of SEQ ID NO:2 or SEQ ID NOS: 3 and 4 in the inclusion bodies;
solubilizing the inclusion bodies in a detergent to release the ScFv antibody;
oxidizing the released ScFv antibody to form disulfide bonds;
removing the detergent;
precipitating the oxidized ScFv antibodies;
dissolving the precipitated ScFv antibodies in a denaturing solution;
immobilizing the ScFv antibodies on a solid support;
renaturing ScFv antibodies on the solid support; and
eluting the ScFv antibodies in biologically active form,
wherein the ScFv antibody binds interferon α-2b.
2. The method of claim 1 , wherein the detergent is N-lauroylsarcosine solution.
3. The method of claim 1 , wherein the oxidation takes place in the presence of a Cu 2+ catalyst.
4. The method of claim 1 , wherein the detergent is removed by butanol extraction.
5. The method of claim 1 , wherein the precipitation is by centrifugation.
6. The method of claim 1 , wherein the denaturing solution is a buffered urea solution.
7. The method of claim 1 , wherein the renaturation is performed with a linear phosphate gradient.
8. The method of claim 1 , wherein the solid support is Ni-NTA agarose.
9. An isolated ScFv 17 protein having the sequence shown in SEQ ID NO: 2.
10. An isolated ScFv17 protein having the amino acid sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:4.