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KMID : 0545120170270050965
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Journal of Microbiology and Biotechnology
2017 Volume.27 No. 5 p.965 ~ p.974
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Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in Escherichia coli and Brevibacillus choshinensis
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Hu Wei
Xiang Jun-Yan
Kong Ping
Liu Ling
Xie Qiuhong
Xiang Hongyu
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Abstract
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The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and response surface methodology, under which the yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature, and particle diameter size were 1.01E-07 M, 55.2 ± 0.3ºC, and 9.388 nm for the scFv expressed by B. choshinensis, and 4.53E-07 M, 52.5 ± 0.3ºC, and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.
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KEYWORD
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Lectin-like oxidized low-density lipoprotein receptor-1, single-chain variable fragment antibody, Brevibacillus choshinensis, Escherichia coli, production methodology
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