• Journal of Life Science
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• v.28 no.12
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• pp.1416-1423
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• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

• Journal of Dairy Science and Biotechnology
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• v.36 no.4
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• pp.208-219
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• 2018

Emergence of drug resistant strains of Salmonella enterica threatens milk processing and related dairy industries, thereby increasing the need for development of new anti-bacterials. Developments of antibacterial drugs are largely aimed to target the bacterial envelope, but screening their efficacy on bacterial envelope is laborious. This study presents a potential biosensor for envelope-specific stress in which a gfp reporter gene fused to spy gene encoding a periplasmic chaperone protein Spy (spheroplast protein y) that can sense envelope stress signals transduced by two major two-component signal transduction systems BaeSR and CpxAR in Salmonella enterica serovars Enteritidis and S. Gallinarum. Using spy-gfp operon fusions in S. Enterititis and S. Gallinarum, we found that spy transcription in both serovars was greatly induced when Salmonella cells were forming the spheroplast and were treated with ethanol or a membrane-disrupting antibiotic polymyxin B. These envelope stress-specific inductions of spy transcription were abrogated in mutant Salmonella lacking either BaeR or CpxR. Results illustrate that induction of Spy expression can be efficiently triggered by two-component signal transduction systems sensing envelope stress conditions, and thereby suggest that monitoring the spy transcription by spy-gfp operon fusions would be helpful to determine if developing antimicrobials can damage envelopes of S. Enteritidis and S. Gallinarum.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1133-1140
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• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.