title: Improvement of Vitreoscilla hemoglobin function by Bacillus licheformis glutamate-specific endopeptidase treatment.
abstract: Vitreoscilla hemoglobin (VHb) was widely used in metabolic engineering to improve oxygen utilization in the low oxygen environment. It is sometimes necessary to remove affinity tags because they may impede functions of target proteins. Here we report an efficient method employing Glutamate-specific endopeptidase from Bacillus licheformis (GSE-BL) to perform the cleavage between VHb and His-tag. The optimal length of GSE-BL treatment was 15min. Results of SDS-PAGE and western blot demonstrated that the His-tag of VHb-His(6) was nearly completely removed, the purity of VHb was enhanced from 74% to 99.5%, and the yield of tagless VHb from VHb-His(6) was 92.2%. Results of CO difference spectrum suggested that tagless VHb was more prone to bind to CO compared with VHb-His(6). It was observed that tagless VHb displayed higher catalase activity than VHb-His(6). The enhancement of welan gum yield was more significant by addition of tagless VHb compared with addition of VHb-His(6). This method can be utilized to mass-produce tagless VHb, thus widening the application of VHb in various industries.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ye
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.forename: Juan
authors.initials: J
authors.lastname: Yang
authors.forename: Haiying
authors.initials: H
authors.lastname: Wang
authors.forename: Xinhui
authors.initials: X
authors.lastname: Wang
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
mesh_terms: D000345:Affinity Labels; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015153:Blotting, Western; D002374:Catalase / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry*; D004789:Enzyme Activation; D057075:Enzyme Assays; D018698:Glutamic Acid / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D011485:Protein Binding; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D054793:Truncated Hemoglobins / Q000737:chemistry / Q000302:isolation & purification*; D020589:Vitreoscilla / Q000737:chemistry*
chemical_list: D000345:Affinity Labels; D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D018698:Glutamic Acid; C068570:welan; D002374:Catalase; D010450:Endopeptidases