title: Enhancement of soluble expression of codon-optimized Thermomicrobium roseum sarcosine oxidase in Escherichia coli via chaperone co-expression.
abstract: The codon-optimized sarcosine oxidase from Thermomicrobium roseum (TrSOX) was successfully expressed in Escherichia coli and its soluble expression was significantly enhanced via the co-expression of chaperones. With the assistance of whole-genome analysis of T. roseum DSM 5159, the sox gene was predicated and its sequence was optimized based on the codon bias of E. coli. The TrSOX gene was successfully constructed in the pET28a plasmid. After induction with IPTG for 8h, SDS-PAGE analysis of crude enzyme solutions showed a significant 43 kDa protein band, indicating SOX was successfully expressed in E. coli. However, the dark band corresponding to the intracellular insoluble fraction indicated that most of TrSOX enzyme existed in the inactive form in "inclusion bodies" owing to the "hot spots" of TrSOX. Furthermore, the co-expression of five different combinations of chaperones indicated that the soluble expression of TrSOX was greatly improved by the co-expression of molecular chaperones GroES-GroEL and DnaK-DnaJ-GrpE-GroES-GroEL. Additionally, the analysis of intramolecular forces indicated that the hydrophobic amino acids, hydrogen bonds, and ionic bonds were favorable for enhancing the interaction and stability of TrSOX secondary structure. This study provides a novel strategy for enhancing the soluble expression of TrSOX in E. coli.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China; State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Tong
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China. Electronic address: fengss@jiangnan.edu.cn.
authors.forename: Shoushuai
authors.initials: S
authors.lastname: Feng
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xin
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China.
authors.forename: Hailin
authors.initials: H
authors.lastname: Yang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China. Electronic address: bioprocessor@aliyun.com.
authors.forename: Wu
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D041862:Chloroflexi / Q000201:enzymology* / Q000235:genetics*; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D050879:Sarcosine Oxidase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D050879:Sarcosine Oxidase
keywords: Co-expression of chaperones; E.coli; Sarcosine oxidase; Soluble expression; Thermomicrobium roseum