title: A combined strategy improves the solubility of aggregation-prone single-chain variable fragment antibodies.
abstract: Recombinant single-chain variable fragment (scFv) antibodies have wide applications in the areas of biotechnology and medicine. However, there is currently no universal expression-purification system for generating different soluble scFvs. In this study, A15 and E34, two genes coding scFvs against human IL-17A, were fused with N-terminal signal peptide sequences pelB or STII, or with highly hydrophilic tags Trx, NusA, or MBP, respectively. These constructs were expressed in Escherichia coli. We found that the scFvs fused with either NusA or MBP showed a higher solubility than fused with signal peptides or Trx. The scFvs were aggregated when the NusA or MBP was removed by thrombin. Interestingly, we observed a reduction of precipitation when the fusion proteins were expressed in Origami B(DE3)pLysS cells but not in BL21(DE3)pLysS. Because cleaving the tags resulted in the aggregation of scFvs, several solubility-enhancing additives were added in the digestion buffer and only L-arginine (Arg) or Tween20 promoted the solubility. After an affinity chromatography, the scFvs were separated from the tags with the purity up to 90%. The final yield of scFvs from the scFv-MBP system was approximately 8.9 mg/L of culture medium and 1.5 mg/g of wet weight cells, which was 1.6-fold higher than the yield from the scFv-NusA system. The obtained scFvs exhibited normal binding affinities and activities after endotoxin removal. In conclusion, we describe a strategy combining the fusion tags, the Escherichia coli with oxidizing bacterial cytoplasm, and the solubility-enhancing additives for expressing and purifying the soluble and functional scFvs.
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.forename: Jing
authors.initials: J
authors.lastname: Xie
authors.forename: Heng
authors.initials: H
authors.lastname: Lin
authors.forename: Su
authors.initials: S
authors.lastname: Mi
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.forename: Fang
authors.initials: F
authors.lastname: Hua
authors.forename: Zhuowei
authors.initials: Z
authors.lastname: Hu
mesh_terms: D002846:Chromatography, Affinity; D006801:Humans; D020381:Interleukin-17 / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D015202:Protein Engineering; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013917:Thrombin / Q000378:metabolism
chemical_list: C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; C508717:IL17A protein, human; D020381:Interleukin-17; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D013917:Thrombin