Expression,
Purification, Characterization of Amphioxus Insulin-like Peptide and
Preparation of Polyclonal Antibody to It
SHEN Lu2, GUO Zhan-Yun1, CHEN Yan1,
LIU Lan-Ying2, FENG You-Min1*
(1State Key Laboratory of Molecular Biology, Shanghai Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
the Chinese Academy of Sciences, Shanghai 200031, China； 2 Life Science College, Jilin University,
Changchun 130061,
China )
Abstract To
elucidate the origination and evolution as well as structure-function
relationship of insulin and IGF-1, the gene of amphioxus insulin-like peptide
(ILP),a common ancestor of insulin and IGF-1, was chemically synthesized and
cloned into the expression vector pVT102-U. In the recombinant ILP, the
C-terminus of B-domain and the N-terminus of A-domain of amphioxus ILP deduced
from cDNA were linked together by a tripeptide, Ala-Ala-Lys, and its B28Arg
residue was also replaced by Lys to facilitate its conversion to a double-chain
form by Lys-C cleavage. The expression vector was transformed into yeast cells
and the recombinant ILP was expressed efficiently. The purified single-chain
and double-chain ILP was obtained by fermentation, purification and enzymatic
cleavage. Molecular weight measurement and amino acid composition analysis
showed that the primary structure of ILP was correct. Circular dichroism
analysis showed that the secondary and tertiary structure of double-chain ILP
was similar to that of insulin, while double-chain ILP had no measurable
insulin activity in insulin receptor binding assay. Additionally, New Zealand
Rabbits were immunized with single-chain ILP and high titre polyclonal antibody
was obtained. This work was important for further investigating the molecular
evolution of insulin and IGF-1 and the distribution and existence of ILP in
amphioxus.
Key words insulin； IGF-1； insulin-like peptide； amphioxus； expression
*Corresponding author： Tel, 86-21-64374430； Fax, 86-21-64338357； e-mail, [email protected]