Biosynthesis and periplasmic segregation of human proinsulin in Escherichia coli.
نویسندگان
چکیده
A plasmid containing human preproinsulin cDNA inserted into the endonuclease Pst I site of the ampicillinase gene of plasmid pBR322 was modified by excision of large portions of the ampicillinase-coding region to produce a variety of gene fusion combinations, many of which generated proteins detectable with antisera to insulin or human C peptide. In one case a perfect hybrid of the NH2-terminal half of the leader sequence of ampicillinase (residues -23 to -12) with the human preproinsulin prepeptide beginning at residue -13 was formed; the result was the synthesis and secretion of human proinsulin into the periplasmic space. We have characterized this protein immunologically and also by labeling it biosynthetically or by iodination followed by immunoprecipitation and automated amino acid sequence analysis. It contains the A and B chain regions of insulin as well as specific human C peptide immunodeterminants and is convertible to an insulin-like component by tryptic digestion. These results demonstrate that human proinsulin can be produced by bacteria and that this biosynthetic approach should prove feasible for the production of adequate amounts of human proinsulin for a variety of clinical studies and human insulin for therapeutic purposes.
منابع مشابه
Comparison of T7- and Lac-Based Systems for the Periplasmic Expression of Human Granulocyte Macrophage Colony Stimulating Factor in Escherichia coli
متن کامل
CONSTRUCTION OF RECOMBINANT PLASMIDS FOR PERIPLASMIC EXPRESSION OF HUMAN GROWTH HORMONE IN ESCHERICHIA COLI UNDER T7 AND LAC PROMOTERS
In order to study the periplasmic expression of human growth hormone (hGH) in Escherichia coli, the related cDNA was inserted in two expression plasmids carrying pelB signal peptide, one with lac bacterial promoter and the other with a bacteriophage T7-based promoter. The recombinant plasmids were moved to TG1 and BL21 strains of E. coli, respectively. To induce the expression systems, IPTG and...
متن کاملOverexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli
Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks and bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon ...
متن کاملOverexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli
Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks and bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon ...
متن کاملPeriplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences
Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful andmost of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role ofnative and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli,the lipase gene (btl2) was cloned downstream of the native ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 78 9 شماره
صفحات -
تاریخ انتشار 1981