[Purification and activity assay of HSA-AX15 (R13K) fusion protein expressed in Pichia pastoris].
To increase the in vivo half-life of human CNTF mutein AX15 (R13K), HSA-AX15 (R13K) fusion protein was constructed by the fusion of the C-terminus of HSA to the N-terminus of AX15 (R13K) via an 11 amino acids linker. HSA-AX15 (R13K) fusion protein was purified to homogeneity by cation exchange chromatography, reverse phase chromatography and gel filtration after expressed in pichia pastoris. TF-1 cell survival bioassay showed the biological activity of AX15 (R13K) was not affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8 mg/kg HSA-AX15 (R13K) fusion protein could produce more potent anti-obesity effects on KM mice than daily injection of 1.6 mg/kg AX15 (R13K). The long-acting form of hCNTF variant has the potential to reduce discomfort by requiring fewer injections and to minimize the side-effects by decreasing the dosage and fluctuation of plasma concentration, and thus has superior clinical application.
Zhao
Hong-Liang
HL
Beijing Institute of Biotechnology, Beijing 100071, China.
Xue
Chong
C
Xiong
Xiang-Hua
XH
Zhang
Wei
W
Yang
Bing-Fen
BF
Liu
Zhi-Min
ZM
D000818:Animals; D020934:Ciliary Neurotrophic Factor / Q000235:genetics*; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012709:Serum Albumin / Q000235:genetics*
D020934:Ciliary Neurotrophic Factor; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin