Journal
LETTERS IN APPLIED MICROBIOLOGY
Volume 68, Issue 3, Pages 241-247Publisher
WILEY
DOI: 10.1111/lam.13110
Keywords
aflatoxin B-1; Escherichia coli; extracellular production; single-chain variable fragment; Triton X-100
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Funding
- Basic Science Research Program through the National Research Foundation of Korea (NRF) - Ministry of Science and ICT [2017R1A2B4004782]
- National Research Foundation of Korea [2017R1A2B4004782] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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scFv-BM3 is a single-chain variable fragment (scFv) against aflatoxin B-1 (AFB(1)) 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 AFB(1), 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 AFB(1). 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 beta-sheet structure and adopted a proper conformation to bind AFB(1) with high affinity and specificity in various biophysical and biochemical analyses.
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