Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in Escherichia coli.
The immunocytokine scFvMEL/TNF is a fusion protein composed of tumor necrosis factor (TNF-alpha) and a single-chain Fv antibody scFvMEL targeting the melanoma-associated gp240 antigen. The fusion protein containing thioredoxin and a hexa-histidine tag was expressed in two Escherichia coli host cells, AD494 (DE3) pLysS and T7 Express I (q). The cell growth and expression level of target protein, His-tagged scFvMEL/TNF, were highly dependent on the induction temperature, inducer types and host strains. The ratio of insoluble to soluble target proteins was found to be controllable and could be minimized using cold shock conditions at less than 18 degrees C. The total productivity of soluble target protein was further improved by high cell density cultivation using a DO-STAT feeding strategy. The scFvMEL/TNF purified under their conditions was specifically cytotoxic to gp240-antigen positive melanoma A375-M cells as previously described.
Kim
Sehoon
S
Department of Experimental Therapeutics, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Cheung
Lawrence H
LH
Zhang
Weihe
W
Rosenblum
Michael G
MG
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007785:Lactose / Q000494:pharmacology; D008545:Melanoma / Q000276:immunology / Q000473:pathology; D011509:Proteoglycans / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D007128:Immunoglobulin Fragments; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C094441:gp240 glycoprotein, human; D007785:Lactose