Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris.
The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.
Laboratory of Genome Engineering, Beijing Institute of Basic Medical Sciences, Beijing, China.
Wen-Rong
WR
Xia
Wen-Liang
WL
Fu
Ling
L
Cai
Xin
X
Cai
Yuan-Yuan
YY
Wang
Min-Ji
MJ
Zou
Dong-Gang
DG
Xu
D000818:Animals; D001681:Biological Assay; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply* / Q000187:drug effects; D002845:Chromatography; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D014170:Transformation, Genetic
D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C046366:angiogenin; D012259:Ribonuclease, Pancreatic