Preparation of Polyacrylamide
Gel Electrophoresis for Human Chorionic Gonadotropin Chimeric Peptide 12
Expressed in E. coli
ZOU Yong-Shui, XU Wan-Xiang*,
HE Yuan, SUN Zhi-Da£¬ XUE Xiao-Lin
( National Laboratory of Contraceptives and Devices Research, Shanghai
Institute of
Planned Parendhood Research, Shanghai 200032, China )
Abstract In recent
years, development of chimeric peptide (CP) immunogens is a trend in the
vaccinological field. The CPs contain a B cell epitope(s) of target antigen and
a ¡°promiscuous¡± self - or foreign- T cell epitope(s). However, such constructed
CPs were all expressed in prokaryotic or eukaryotic systems at lower levels. To
purify the human chorionic gonadotropin (hCG) CP12 expressed in E. coli
at the level of about 1% of the total cell proteins, an improved method of
preparative gel polyacrylamide gel electrophoresis (PAGE) was developed. The important
improvement to routine preparative PAGE involves: (1) running reversed
electrophoresis by rearranging the gel- carrying plate when the bromophenol
blue band arrived at 1-1.5 centimeter from the bottom of the gel; (2) making a
collecting trough between the gel and a dialytic membrane that was used to
isolate the upper tank buffer. About 8 fractions were collected at regular
intervals of 15 minutes after bromophenol blue running out of gel. And then 0.2
ml was taken from each fraction and the protein was precipitated by
sequentially adding trichloroacetic acid and acetone. Each sample was dissolved
in 20 ¦Ìl sample buffer and analyzed and identified by SDS-PAGE and Western
blotting. As a result, the hCG CP12 expression product with 95% relative homogeneity
was harvested at a 50-100 ¦Ìg level after a single-step purification of this
preparative PAGE, with respect to the sample which contained 3-4 mg of cell
proteins.
Key words human chorionic gonadotropin; bio-engineered
chimeric peptide; preparative PAGE; purification
*Corresponding author£ºTel, 86-21-64042641; Fax,
86-21-64677609; e-mail, [email protected]