Details

Autor(en) / Beteiligte

• Ya-ping HE Ya NI Ai-zhen HONG Li-wen SONG Yu-bao WANG Si-chang CHOW Yu-ying YUAN Qi-xian SHI Elvira Hinsch Klaus-Dieter Hinsch Wan-xiang XU

Titel

High-level Expression and Purification of Human Zona Pellucida huZP3a^22-176 and huZP3b^177-348 Peptides in Escherichia coli

Ist Teil von

• Journal of reproduction and contraception, 2005, Vol.16 (1), p.1-10

Erscheinungsjahr

2005

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a higher level Methods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method. The pBV221 plasmid was used to construct thermo-inducible recombinant expression vector. Purification of two target expression products employed an improved method of preparative gel polyacrylamide gel electrophoresis. Results Two polypeptides of recombinant huZP3a （rhuZP3a） and recombinant huZP3b （rhuZP3b） were all expressed respectively in an E. coli BL21（DE3）pLysS strain at a higher level, which were recognized by two specific polyclonal antisera in Western blotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3b respectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3b with more than 95% relative homogeneity were harvested from 1 L culture respectively. Conclusion The availability of two rhuZP3 polypeptides will help in detecting the immunogenicities of rhuZP3a and rhuZP3b through animal experiments and confirming the function domain of non-glycosylated huZP3 to induce acrosome reaction in vitro.