Production of a biologically active recombinant marsupial growth factor using the methylotrophic yeast Pichia pastoris.
The cytokine stem cell factor (SCF) and its interaction with its receptor c-kit plays an important role in the development of germ cells in eutherians. To investigate the putative roles of the SCF/c-kit system in marsupials, recombinant Australian brushtail possum (Trichosurus vulpecula) SCF was purified after secretion by the methylotrophic yeast Pichia pastoris. The purification procedure utilized Ni2+ affinity chromatography with a poly-histidine tag engineered onto the C-terminus of the recombinant SCE The recombinant possum SCF had a molecular weight of 48 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was biologically active with respect to its ability to maintain and induce proliferation of marsupial primordial germ cells in vitro. Furthermore, the recombinant possum SCF stimulated proliferation of the cell line TF1 and this bioactivity could be inhibited using an antibody directed against recombinant mouse SCF. This source of biologically active marsupial SCF may prove useful in future studies of marsupial development.
Fidler
Andrew E
AE
AgResearch Wallaceville, Upper Hutt, New Zealand.
Western
Andrea H
AH
Griffith
Nicole
N
Selwood
Lynne
L
Stent
Vicki
V
McNatty
Kenneth P
KP
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009532:Nickel; D009893:Opossums / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019089:Stem Cell Factor / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D019089:Stem Cell Factor; D009532:Nickel