title: High level soluble production of functional ribonuclease inhibitor in Escherichia coli by fusing it to soluble partners.
abstract: Ribonuclease inhibitor (RI) is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases which inhibits ribonucleolytic activity. Expression of recombinant RI is extremely difficult to reach high levels in soluble form in the cytoplasm of Escherichia coli. Here, we utilized five N-terminal fusion partners to improve the soluble expression of RI. Among these five fusion partners which have been screened, maltose-binding protein (MBP), N-utilization substance A (NusA) and translation initiation factor 2 domain I (IF2) have greatly improved the soluble expression level of recombinant murine RI under the drive of T7 promoter, while glutathione S-transferase (GST) and small ubiquitin modifying protein (SUMO) were much less efficient. All these RI-fusion proteins remained to be highly active in inhibiting RNase A activity. Furthermore, all fusion tags can be efficiently removed by enterokinase digestion to generate native RI which results the highest yield to date (>30mg of native RI per liter culture). And a convenient two-step immobilized metal affinity chromatography (IMAC) method has been implemented in our study, comparing with the traditional RNase A affinity chromatography method.
authors.affiliation: Department of Nuclear Medicine, Nanjing University, Nanjing 210008, People's Republic of China.
authors.forename: Wanhua
authors.initials: W
authors.lastname: Guo
authors.forename: Lin
authors.initials: L
authors.lastname: Cao
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Jia
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
authors.forename: Teng
authors.initials: T
authors.lastname: Li
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lu
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
mesh_terms: D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D039881:Prokaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D035602:Transcriptional Elongation Factors
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D010445:Peptide Elongation Factors; D039881:Prokaryotic Initiation Factor-2; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D005982:Glutathione Transferase; D012259:Ribonuclease, Pancreatic; D004765:Enteropeptidase