Secretory Expression of the Active Domain of Murine RANKL in P.pastoris

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Chinese Journal of Biochemistry and Molecular Biology ›› 2003, Vol. 19 ›› Issue (04) : 451-456.

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Secretory Expression of the Active Domain of Murine RANKL in P.pastoris

WANG Bao\|li, GUO Gang, LIANG Dong\|chun, ZHAO Xue\|qin,ZHANG Jing\|yu, QIU Ming\|cai 

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Abstract

Receptor activator of nuclear factor κB ligand (RANKL) is a key regulator of formation and activation of osteoclasts.Total RNA was extracted from mouse bone tissue using the modified acid guanidinium thiocyanate phenol chloroform method. The cDNA encoding the active domain of murine RANKL (amino acid 158—316) was amplified through RT PCR, then inserted into the Xho Ⅰ and Not Ⅰ sites of pPIC9, a secretory expression vector. The recombinant plasmid was transformed into GS115, a Pichia host strain. Hundreds of His + Mut + genotype colonies were screened for expression induced with methanol. Total protein from the recombinant cells presented an additional thick band with a molecular weight of about 26 kD on Tricine SDS PAGE gel compared with that from the induced cells transformed with the void plasmid pPIC9. The protein could be recognized, through Western blot analysis by the polyclonal antibody against RANKL. The recombinant protein was purified to homogeneity through a series of methods including salting out with ammonium sulfate, ion exchange chromatography. The yield of the secreted product of interest was about 11 mg/L, assessed by BCA protein assay. It was confirmed that the recombinant protein was biologically active in that it stimulated the formation of osteoclast like cells from bone marrow cells.

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receptor activator of nuclear factor κB ligand / Pichia pastoris / secretory expression

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Received	Revised	Published
2003-08-20	2003-08-20	2003-08-20
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2003-08-20

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