title: Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners.
abstract: Heparinase I (HepA) was originally isolated from Flavobacterium heparinum (F. heparinum) and specifically cleaves heparin/heparan sulfate in a site-dependent manner, showing great promise for producing low molecular weight heparin (LMWH). However, expressing recombinant HepA is extremely difficult in Escherichia coli because it suffers from low yields, insufficient purity and insolubility. In this paper, we systematically cloned and fused the HepA gene to the C-terminus of five soluble partners, including translation initiation factor 2 domain I (IF2), glutathione S-transferase (GST), maltose-binding protein (MBP), small ubiquitin modifying protein (SUMO) and N-utilization substance A (NusA), to screen for their abilities to improve the solubility of recombinant HepA when expressed in E. coli. A convenient two-step immobilized metal affinity chromatography (IMAC) method was utilized to purify these fused HepA hybrids. We show that, except for NusA, the fusion partners dramatically improved the soluble expression of recombinant HepA, with IF2-HepA and SUMO-HepA creating almost completely soluble HepA (98% and 94% of expressed HepA fusions are soluble, respectively), which is the highest yield rate published to the best of our knowledge. Moreover, all of the fusion proteins show comparable biological activity to their unfused counterparts and could be used directly without removing the fusion tags. Together, our results provide a viable option to produce large amounts of soluble and active recombinant HepA for manufacturing.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
authors.forename: Lin
authors.initials: L
authors.lastname: Cao
authors.forename: Wanhua
authors.initials: W
authors.lastname: Guo
authors.forename: Ruoxi
authors.initials: R
authors.lastname: Yuan
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Jia
authors.forename: Kehe
authors.initials: K
authors.lastname: Huang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D019763:Heparin Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D004765:Enteropeptidase; D019763:Heparin Lyase