大肠杆菌
片段(逻辑)
细胞外
微生物学
黄曲霉毒素
单链可变片段
生物
肠杆菌科
化学
细菌
分子生物学
遗传学
重组DNA
食品科学
基因
程序设计语言
计算机科学
作者
K.-I. Na,S.-J. Kim,Duk-Ho Choi,Won‐Ki Min,Sung‐Gun Kim,Jin‐Ho Seo
摘要
scFv-BM3 is a single-chain variable fragment (scFv) against aflatoxin B1 (AFB1 ) engineered by affinity maturation and site-directed mutagenesis, and thus has a 31-fold higher affinity than its wild-type. To apply scFv-BM3 to immunological detection of AFB1 , periplasmic expression in Escherichia coli was attempted to produce a functional form of scFv-BM3. scFv-BM3 accumulated as inactive aggregates in the cells. However, it was found that scFv-BM3 secreted into the culture medium had binding activity to AFB1 . Expression conditions for scFv-BM3 were further manipulated to enhance secretion into the culture medium. This extracellular secretion of functional scFv-BM3 was significantly improved by supplementation with Triton X-100 and optimization of expression conditions. The scFv-BM3 purified from the culture medium exhibited a typical antiparallel β-sheet structure and adopted a proper conformation to bind AFB1 with high affinity and specificity in various biophysical and biochemical analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: Single-chain variable fragments (scFvs) are recombinant antibodies that are difficult to produce as a functional form in Escherichia coli. This study demonstrates the production of functional scFvs against aflatoxin B1 (AFB1 ) (scFv-BM3) using Escherichia coli by extracellular secretion. While periplasmic expression of scFv-BM3 resulted in formation of inactive aggregates in E. coli, the scFv-BM3 secreted into the culture medium adopted a properly folded structure for specific binding to AFB1 . This study promotes the application of functional scFv-BM3 to the immunological detection of AFB1 in biotechnology fields.
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