title: Co-expression of a heat shock transcription factor to improve conformational quality of recombinant protein in Escherichia coli.
abstract: A co-expression system was established in Escherichia coli for enhancing the cellular expression of heat shock transcription factor, sigma 32 (σ(32)). A Shine-Dalgarno sequence and the rpoH gene of E. coli, which encodes σ(32), were cloned into a bacterial plasmid containing a gene fusion encoding a doubly tagged N-acetyl-d-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R). After the IPTG induction, a substantially higher level of sigma 32 was observed up to 3 h in the co-expression cells, but an enhancement in the solubility of target protein was manifest only in the first hour. Nevertheless, the co-expression of sigma 32 led to higher level of Neu5Ac aldolase enzymatic activity in both the soluble and insoluble (inclusion body) fractions. The Neu5Ac aldolase activity of the supernatant from the lysate of cells co-expressing GST-Neu5Ac aldolase-5R and recombinant σ(32) was 3.4-fold higher at 3 h postinduction than that in cells overexpressing GST-Neu5Ac aldolase-5R in the absence of recombinantly expressed σ(32). The results of acrylamide quenching indicated that the conformational quality of the fusion protein was improved by the co-expression of recombinant σ(32). Thus, the increased level of intracellular σ(32) might have created favorable conditions for the proper folding of recombinant proteins through the cooperative effects of chaperones/heat shock proteins expressed by the E. coli host, which resulted in smaller inclusion bodies, improved conformational quality and a higher specific activity of the overexpressed GST-Neu5Ac aldolase-5R protein.
authors.affiliation: Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan.
authors.forename: Shao-Yen
authors.initials: SY
authors.lastname: Hsu
authors.affiliation: Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan.
authors.forename: Yu-Sheng
authors.initials: YS
authors.lastname: Lin
authors.affiliation: Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan.
authors.forename: Shu-Jyuan
authors.initials: SJ
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan. Electronic address: chmwcl@ccu.edu.tw.
authors.forename: Wen-Chien
authors.initials: WC
authors.lastname: Lee
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D012995:Solubility
chemical_list: D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D007544:Isopropyl Thiogalactoside; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase
keywords: Chaperones; Conformational quality; Escherichia coli; Fusion protein; Heat shock proteins; Inclusion body; Sigma factor 32