胰岛素
重组DNA
胰岛素原
色谱法
化学
消化(炼金术)
发酵
门冬氨酸胰岛素
高效液相色谱法
人胰岛素
大肠杆菌
生物化学
生物
内分泌学
餐后
基因
作者
Satish Babu Kaki,A. Naga Prasad,Anjani Devi Chintagunta,Vijaya Ramu Dirisala,N. S. Sampath Kumar,S. J. K. Naidu,B. Ramesh
标识
DOI:10.1007/s40995-022-01269-7
摘要
Insulin is a well-characterized peptide produced by recombinant DNA technology that is widely used in maintaining blood glucose level in the diabetic patients. In this study, we have used vector pET-9a to express the human insulin in E. coli BL-21 DE3. The recombinant E. coli cells were cultured by fed-batch fermentation (20.0 L) process which resulted in a dry cell mass of 20 g/L. Pro-Insulin was expressed as inclusion bodies (IBs) and it was 20% of the total protein measured by densitometry. The IBs were then solubilized and refolded to get the native biological structure. The proinsulin form has been converted into active insulin by enzymatic digestion using trypsin and carboxypeptidase-B. The active insulin was purified using ion-exchange and reverse phase HPLC. The final purified insulin has achieved a purity of 98% with Des-Insulin impurity ≤ 0.5%, when analyzed in analytical HPLC as per the United States Pharmacopiea (USP) method. This optimized process of recombinant human insulin may be used as a model for insulin analogues such as glargine, aspart, lispro etc.
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