Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli.
- Author:
Byung Ung HUR
1
;
Hyo Jung CHOI
;
Jae Bong YOON
;
Sang Hoon CHA
Author Information
- Publication Type:Original Article
- Keywords: Fv; Leucine zipper; Recombinant antibody; Alkaline phosphatase; DsbC
- MeSH: Alkaline Phosphatase; Antibodies; Clone Cells; Cytoplasm; DNA; Escherichia; Escherichia coli; Immunoglobulin Fragments; Indicators and Reagents; Leucine Zippers; Oxidoreductases; Pathology, Molecular; Pyruvic Acid; Sprains and Strains
- From:Immune Network 2010;10(2):35-45
- CountryRepublic of Korea
- Language:English
- Abstract: BACKGROUND: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. METHODS: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize V(H) and V(L) fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. RESULTS: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of V(H) or V(L) domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of V(H) of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. CONCLUSION: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.
