[Preparation of polyacrylamide gel electrophoresis for human chorionic gonadotropin chimeric peptide 12 expressed in E. coli].
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 microL 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 microgram level after a single-step purification of this preparative PAGE, with respect to the sample which contained 3-4 mg of cell proteins.
Zou
Yong-Shui
YS
National Laboratory of Contraceptives and Devices Research, Shanghai Institute of Planned Parendhood Research, Shanghai 200032, China. xuwx@sippr.stc.sh.cn
Xu
Wan-Xiang
WX
He
Yuan
Y
Sun
Zhi-Da
ZD
Xue
Xiao-Lin
XL
D015153:Blotting, Western / Q000379:methods; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D006063:Chorionic Gonadotropin; D011993:Recombinant Fusion Proteins