title: Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.
abstract: T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.
authors.affiliation: Department of Applied Biochemistry, Tokai University School of Engineering.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Yuasa
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Koyama
authors.forename: Yoko
authors.initials: Y
authors.lastname: Fujita-Yamaguchi
mesh_terms: D000906:Antibodies / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D003199:Computer Systems; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017510:Protein Folding; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000906:Antibodies; D017931:DNA Primers; D057127:Single-Chain Antibodies; C025991:Thomsen-Friedenreich antibodies