Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system.
Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg/L. rhFL was purified to about 95% purity with overnight dialysis, filtration and an anion-exchange step. Further purification steps employing Sephacryl S-200 and reverse-phase HPLC raised the purity to over 99%. The purified rhFL possessed correct N-terminal amino acid sequence and positive Western blotting bands. SDS-PAGE and mass spectrometry analysis showed molecular weight of rhFL was about 21 and 34 kDa, suggesting that rhFL was glycosylated. The result of capillary electrophoresis showed that its pI is 3.12-4.72. Endo H deglycosylation analysis indicated that there was O-glycosylation besides N-glycosylation in rhFL secreted from P. pastoris. Bioactivity assay showed that the purified rhFL had dose-dependent expansion activity on bone marrow nucleated cells.
Zhang
Yan-Li
YL
National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China. zh_yanli@hotmail.com
Chen
Song-Sen
SS
Yang
Ke-Gong
KG
Su
Lin
L
Deng
Yan-Chun
YC
Liu
Chang-Zheng
CZ
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002467:Cell Nucleus / Q000502:physiology; D006133:Growth Substances / Q000502:physiology; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D051379:Mice; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D006133:Growth Substances; D008024:Ligands; D008565:Membrane Proteins; D011994:Recombinant Proteins; C084881:flt3 ligand protein