Influence of growth limiting factors on recombinant growth hormone expression in E.coli
Data
2005Autorius
Morkūnas, Mindaugas
Ališauskaitė, Saulė
Sereikaitė, Jolanta
Bumelis, Vladas Algirdas
Metaduomenys
Rodyti detalų aprašąSantrauka
High level expression of recombinant growth hormones in E.coli faces common problems such as protein aggregation and inclusion body formation. Discussions are raised whether it is more benefi- cial to obtain soluble protein but to loose expression rates. Here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. Experiments were performed on bovine, chicken and mink growth hormones. Expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. Another approach to the problem was performed through genetic manipulation. We changed strong T7 promoter to E. coli promoter consensus sequence thus reducing expression rate. Recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levels – several (2–8) percent of total intracellular protein. We developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation.