Co-expression and purification of recombinant human insulin-like growth factor II and insulin-like growth factor binding protein-6 in Pichia pastoris yeast.
For the preparation of the complex of IGF-II and IGFBP-6, a co-expression vector containing two copies of human IGF-II and IGFBP-6 expression cassette was constructed with alcohol oxidase (AOX1) promoter and secretion signal sequence of alpha-factor, and transformed to Pichia pastoris yeast. Through a purification procedure involving anion-exchange chromatography and gel filtration, a complex of IGF-II with IGFBP-6 was obtained. An additional C-terminal sequence of IGFBP-6 (CS-BP6) was found to be bound to this complex. Dynamic light scattering showed that this complex was very stable and homogenous in solution. Western blotting based on non-reducing Tricine-SDS-PAGE indicated that IGF-II expression coupled with IGFBP-6 might significantly avoid the mispairing of disulfide bonds compared with the IGF-II expressed alone.
Zhou
Huina
H
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Chen
Zhaoli
Z
Chen
Hong
H
Li
Shuxing
S
Huang
Bingren
B
Bi
Ruchang
R
D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
C035170:IGF2 protein, human; D018976:Insulin-Like Growth Factor Binding Protein 6; D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II