The effects of induction conditions on production of a soluble anti-tumor sFv in Escherichia coli.
CC49 is a second generation monoclonal antibody (mAb) with high affinity to a pancarcinoma antigen, TAG-72. A single-chain Fv (sFv) of CC49 may have a role in managing human carcinomas. Most reported sFvs have been expressed as insoluble products that must be solubilized and renatured. Soluble sFv expression is advantageous as activity can be assayed directly from the periplasmic fraction. Also, gene-level immunoconjugates may not be amenable to refolding protocols. Using a vector that carries the tac promoter and omp A signal, we have examined the effects of four variables on the expression and accumulation of soluble CC49 sFv: (i) linker sequence joining VL and VH, (ii) isopropylthio-beta-D-galactoside concentration for induction, (iii) temperature, and (iv) the addition of non-metabolizable sugars to the medium. We have been able to demonstrate, using rapidly prepared periplasmic extracts, that the yield of soluble sFv improves by the addition of 0.4 M sucrose to the medium and by inducing expression with a very low concentration of IPTG (0.02-0.03 mM). Under these induction conditions periplasmic extracts demonstrate increased expression of the sFv, as shown by the larger amount of a 27 kDa band on SDS-polyacrylamide gel, and an increased ability to inhibit binding of the mAb CC49 to immobilized tumor extracts.
Sawyer
J R
JR
Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Schlom
J
J
Kashmiri
S V
SV
D000595:Amino Acid Sequence; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D000951:Antigens, Neoplasm / Q000276:immunology*; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006023:Glycoproteins / Q000276:immunology*; D006358:Hot Temperature; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D013395:Sucrose / Q000494:pharmacology
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; C091202:B72.3 antibody; D017931:DNA Primers; D006023:Glycoproteins; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; C047052:tumor-associated antigen 72; D007544:Isopropyl Thiogalactoside; D013395:Sucrose