title: Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli.
abstract: In our previous studies, the recombinant gonadotropin-releasing hormone (GnRH) peptide was constructed into a T7 RNA polymerase-based expression system. The recombinant gene encoding GnRH3-hinge-MVP, which contained three repeated GnRH units, a fragment of hinge region (225-232/225'-232'), and a T cell epitope of measles virus protein, was cloned into Escherichia coli BL21 harboring pED-GnRH3. The high activity of T7 RNA polymerase could make the expression system very powerful for high-level expression of the recombinant protein. However, during the large-scale production of recombinant protein, the productivity of the fermentation process was directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, the time of induction by lactose, and the number of successive cultures. The results indicate that the plasmid instability may be caused by a loss of plasmid rather than structural change. However, to go down to future generations, engineered bacteria have the stability of plasmid and protein yield to a large extent. The amount of the fusion protein was also up to 40% of the total cell protein after the 50th generation. These data would be useful for the industrial production of the recombinant GnRH vaccine.
authors.affiliation: School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing, People's Republic of China. jinshuuxu@yahoo.com
authors.forename: Jinshu
authors.initials: J
authors.lastname: Xu
authors.forename: Wenjia
authors.initials: W
authors.lastname: Li
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.forename: Yin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhu
authors.forename: Jingjing
authors.initials: J
authors.lastname: Liu
authors.forename: Zhuoyi
authors.initials: Z
authors.lastname: Hu
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000917:Antibody Formation; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D018984:Epitopes, T-Lymphocyte / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015870:Gene Expression; D005786:Gene Expression Regulation; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D007612:Kanamycin / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D008459:Measles virus / Q000235:genetics; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012641:Selection, Genetic; D013997:Time Factors; D014614:Vaccines, Synthetic / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D018984:Epitopes, T-Lymphocyte; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D014764:Viral Proteins; D007987:Gonadotropin-Releasing Hormone; D007612:Kanamycin; D007785:Lactose