Adiponectin Gene Cloning and Its Expression in Insect Cell Expression System

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 36권 / 3호
ㆍ저자명 : In Suh Yuh, Lewis G. Sheffield

목차

ABSTRACT INTRODUCTION MATERIALS AND METHODS Mouse Fibroblasts Differentiation Ooning of the Full-Length Adiponectin: Cell Culture and Transfection Precipitation of the Recombinant Adiponectin by Polyethylene Glycol (PEG): Wheat Germ Agglutination (WGA) Purification Ni-NTA Column Chromatography Q-Sepharose Column Purification Testing for Adiponectin Expression RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

영어 초록

This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (～35 kDa), dimer (～70 kDa), trimer (～105 kDa) and hexamer (～210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (～300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher’s study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (β- MeOH), anionic detergent (SDS) and heat (95℃) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

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