title: Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector.
abstract: Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed "pCold-PDI" vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains.
authors.affiliation: Structural Glycobiology Team, Systems Glycobiology Research Group, Chemical Biology Department, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 3510198, Japan.
authors.forename: Ganesh P
authors.initials: GP
authors.lastname: Subedi
authors.forename: Tadashi
authors.initials: T
authors.lastname: Satoh
authors.forename: Shinya
authors.initials: S
authors.lastname: Hanashima
authors.forename: Akemi
authors.initials: A
authors.lastname: Ikeda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakada
authors.forename: Reiko
authors.initials: R
authors.lastname: Sato
authors.forename: Mamoru
authors.initials: M
authors.lastname: Mizuno
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Yuasa
authors.forename: Yoko
authors.initials: Y
authors.lastname: Fujita-Yamaguchi
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaguchi
mesh_terms: D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology*; D002241:Carbohydrates / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C040286:Tn antigen; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases