Expression, purification, and characterization of recombinant human neurturin secreted from the yeast Pichia pastoris.
Neurturin (NTN), a potent neurotrophic factor acting specifically on dopaminergic neurons, is comprised of 102 amino acids as a mature protein. We artificially synthesized a gene for mature human NTN (hNTN) using codons preferred by the yeast Pichia pastoris. This synthesized gene, fused in frame with sequences encoding the alpha-factor signal peptide gene from Saccharomyces cerevisiae was cloned into P. pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-hNTN was then transformed into the yeast and stable multicopy recombinant P. pastoris strains were selected by G418 resistance. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hNTN, a 16kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using CM-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Bioactivity of the recombinant hNTN was confirmed by the ability of the protein to stimulate growth of nerve fibers from the dorsal root ganglia of chick embryos in vitro.
Li
Hongjun
H
Department of Molecular Biology, Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming, Yunnan 650118, People's Republic of China. lihj6912@hotmail.com
Ma
Yanbing
Y
Su
Ting
T
Che
Yanchun
Y
Dai
Changbai
C
Sun
Maosheng
M
D000818:Animals; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D009414:Nerve Growth Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D051101:Neurturin; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*
C495053:NRTN protein, human; D009414:Nerve Growth Factors; D051101:Neurturin; D011993:Recombinant Fusion Proteins