title: [Activity and quality comparison of the engineered protein Staphylococcus aureus alpha-hemolysin purified with gel filtration chromatography and Ni-NTA].
abstract: The alpha-hemolysin protein of Staphylococcus aureus, which was expressed in Escherichia coli BL21 (DE3) with recombinant pET32a(+)-alpha-HL plasmid, was purified with gel filtration chromatography (GFC) and Ni-NTA spin columns. The quality and biological characteristic were compared. First, the purified products were analyzed with SDS-PAGE, and the expected protein band was with a molecular mass of 53 kD. Second, protein concentration was determined by the method of Bradford, and the median hemolytic dose potency (HD50) was finally analyzed with rabbit erythrocyte. The protein purified with GFC was 0.337 mg/mL, its hemolysis activity was 1519 HU/mg, and hemolysin yield was 14.04%. Meanwhile, the protein purified with the Ni-NTA Spin Columns was 0.35 mg/mL, its hemolysis activity was 1463 HU/mg, and hemolysin yield was 17.5%, respectively. The results showed that there is no significant difference in the quality, hemolysis activity and yield of the recombinant proteins purified with Ni-NTA spin columns and GFC.
authors.affiliation: Cow Research Center, Agricultural Science Academy of Shandong Province, Jinan 250100, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhang
authors.forename: Hongjun
authors.initials: H
authors.lastname: Yang
authors.forename: Changfa
authors.initials: C
authors.lastname: Wang
authors.forename: Hongbin
authors.initials: H
authors.lastname: He
authors.forename: Weiming
authors.initials: W
authors.lastname: Ma
authors.forename: Shaohua
authors.initials: S
authors.lastname: Yang
mesh_terms: D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000235:genetics / Q000302:isolation & purification*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism
chemical_list: D006460:Hemolysin Proteins; D009942:Organometallic Compounds; D011994:Recombinant Proteins; C088321:nickel nitrilotriacetic acid; D009571:Nitrilotriacetic Acid