A Chimera Polypeptide
with Active Sites of HWTX- I and AAI
WANG Xian-Chun, LIANG Song-Ping
( College of Life Science, Hunan Normal University, Changsha 410081, China
)
LUO Ze-Min
( Department of Biological Technology, Hunan Agricultural University,
Changsha 410128, China )
Abstract The functional
amino acid sequence and its neighbouring fragments in the molecule of Amaranth
α-amylase inhibitor isolated from seeds of the Mexican crop plant Amaranthus hypochondriacus were
transferred, by solid phase chemical synthesis, into N-terminal region of the
huwentoxin-I(HWTX-I). The synthetic chimera polypeptide was confirmed by Edman
degradation and MALDI-TOF mass spectroscopy. The formation of three disulfide
bonds and special conformation of the synthetic chimera was induced by the
addition of glutathione. Renatured chimera polypeptide was purified by
ion-exchange and reversed phase HPLC. The results showed that the engineered
chimera polypeptide exerted obvious inhibitory activity to α-amylase from
digestive tube of the roach (Periplaneta americana) at pH 5.5
with the concentration of 9.5×10-5 mol/L, and also exerted 36% of
the neurotoxic activity of the natural huwentoxin-I as shown by the experiments
of blockage of the neuromuscular transmission of isolated mouse phrenic
nerve-diaphragm preparations. The experiments demonstrated that the structural
motif of HWTX- I is well promising for protein engineering, and the solid-phase
peptide synthesis is adequate rapid for the engineering of artificially
designed small proteins.
Key words peptide chimera； Huwentoxin-I； amaranth α-amylase inhibitor； protein engineering
Received: October 12,
1999 Accepted: January 14, 2000
This work was supported by a grant from the National High Technology
“863” Programs of China, No. 103-13-01-06
* Corresponding author: Tel, 86-731-8872556; Fax, 86-731-8861304; e-mail, [email protected]