High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli

Abstract

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the highyield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.

A simple strategy to obtain ultra-sensitive single-chain fragment variable antibodies for aflatoxin detection

“…25 Aer expression, the E. coli cells were extracted using ultrasonication and the extracts were checked by SDS-PAGE with Coomassie Brilliant Blue R-250 Staining. As shown in Fig.…”

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

“…Fusion to maltose-binding protein (MBP) has been shown to not only increase solubility of antibody fragments [10,11], but also enhance secretion from periplasm into the culture medium in secretory E. coli strains [10]. MBP fusion [12] as well as thioredoxin [13] and SUMO fusions [14] have also been reported to improve scFv yields in the cytoplasm of redox mutant strains. In some cases yield may also be increased by engineering the amino acid sequence in non-binding regions of the fragment to reduce its aggregation tendency [15].…”

Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode

“…It has also been reported that the addition of some fusion partners such as maltose‐binding protein, small ubiquitin‐related modifier (SUMO) (Ye et al . ) and thioredoxin (Jurado et al . ) to the target antibody fragment could enhance the solubility and the folding quality (Chames et al .…”