In Vitro Amidating
Processing of Products Expressed by Gene Engineering
YANG Yu-Hong, CHEN Hu, YU Chao, ZENG Rong, CUI Da-Fu, WU
Xiang-Fu，
LI Bo-Liang*
( Shanghai Institute of Biochemistry, the Chinese Academy of Sciences,
Shanghai 200031, China )
Abstract To set up an in vitro amidating
system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the
amidating substrate of recombinant rat peptidylglycine α-amidating
monooxygenase (rPAM). First, the mhCT-Gly gene was synthesized and
cloned into a fusion expression vector to get an expression plasmid pGEXCT. The GST-fused mhCT-Gly was
highly expressed in E.coli BL21(DE3)
harboring the pGEXCT, and was
purified rapidly by affinity chromatography. Second, using the method of
ultrafiltration, the rPAM was prepared from the supernatant of cultured
transfectant CHO cells which express rPAM stably. Finally, the in vitro amidating
experiments were carried out using GST-mhCT-Gly as substrate and the prepared
rPAM. The results of dot blot with the specific antibody and of mass spectrum
assay indicated that amidating product GST-hCT-NH2 could be easily
detected. This study provides a useful method for the amidation of recombinant
products in
vitro.
Key words human calcitonin； recombinant expression； peptidylglycine α-amidating
monooxygenase； in vitro amidation
Received:
September 20, 1999 Accepted: december 13, 1999
This work was supported by National High Technology
“863” Programs of China(No. 863-102-11-03-02) and Science and
Technology Commission Foundation of Shanghai City(No. 97xD14022)
* Corresponding author: Tel, 86-21-64374430-288; Fax,
86-21-64338357; e-mail, [email protected]