Expression, Separation And Purification Of Recombinant Human Insulin In E.Coli | Posted on:2009-09-26 | Degree:Master | Type:Thesis | Country:China | Candidate:Y Chen | Full Text:PDF | GTID:2120360245474786 | Subject:Pharmaceutical Engineering | Abstract/Summary: | PDF Full Text Request | In spite of the clinic application of insulin has 80 years' history. Up today Insulin was still far-ranging needed because there are a great numbers of diabetic patients around the world. So expression system and expression strategy of the recombination human insulin were a hot research topics.An over-expression vector pET28a(+)-insulin had been constructed in our lab, which was transferred into E.coli Rosettatm (DE3) for a high-level expression of human insulin. The optimal culture conditions for the genetic engineered E.coli Rosettatm (DE3) were as follows: The bacterium cells were cultured at 37℃, following inducted with 0.5 mmol·L-1 IPTG when the cell density at OD600 reached 0.8. After induction for 4hrs, cells were harvested by centrifugation at 1000×g for 10min at 4℃.The rhpreinsulin protein expressed in E. coli was usually accumulated as inclusion bodies in the bacterium. Inclusion bodies was washed by different washing solutions with 2 mol/L Urea, washing 3 times. After denaturaing, the denatured liquid was added to the renaturation solution. The optimal renatural conditions were as follows: pH9.5, 60 mmol/L glycin, 60 mmol/LDTT, 2mmol·L-1 GSH-0.2mmol·L-1GSSG With this procedure the renatura efficiency is increased to 65%. After renatura, the sample was loaded on a chromatography column DEAE-Sepharose FF and eluted by the buffer containing 0.1mol/L NaCl. The purified rhpreinsulin was cleaved by TPCK-trypsin and carbonpeptidase B. Finally, gel chromatography Sephadex G-25 was performed to obtain the matured rhInsulin which was identified by HPLC.The experiment results provided a good reference for the expression and purification of rhInsulin in E.coli.
| Keywords/Search Tags: | recombinant human preinsulin, E.Coli, fermentation, renaturation, separation and purification | PDF Full Text Request | Related items |
| |
|