Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin

Abstract

Background: Insulin controls hyperglycemia caused by diabetes, and virtually all treatments require exogenous insulin. However, the product's extensive post-translational modifications have hindered the manufacture of recombinant insulin. Result: Here we report a novel production method for a monomeric B22Asp desB30 insulin analog (B22D desB30 insulin). Its precursor, DPIP, is fused to an N-terminal chitin-binding domain and intein self-cleavage tag. The fusion protein is expressed and purified from E. coli and immobilized on chitin resins. DPIP is then released using an optimized pH shift and converted to mature insulin via trypsin digest. The resulting product appears monomeric, > 90% pure and devoid of any exogenous enzyme. Conclusion: Thus, biologically active insulin analog can be efficiently produced in bacteria and potentially applicable in the treatment of human diabetes.

Recombinant production of interleukin-1 receptor antagonist in fusion to albumin binding domain with potential affinity to human serum albumin

“…reported that the best temperature was considered 37 °C among investigated temperatures ranging from 15 to 37 °C for Ssp DnaB mini-intein fused to human GLP-1/7-36 ( 17 ). It seems that lowering pH to even 4.5 can increase the time of column incubation and the temperature of incubation leading to the efficient cleavage of intein 1 ( 18 ). One study performed on the cleavage of a mutated form of insulin from the intein1 reported that pH 4.5 in comparison to pH 8, and the temperature of 25 °C was more efficient in cleavage ratios ( 18 ).…”

“…It seems that lowering pH to even 4.5 can increase the time of column incubation and the temperature of incubation leading to the efficient cleavage of intein 1 ( 18 ). One study performed on the cleavage of a mutated form of insulin from the intein1 reported that pH 4.5 in comparison to pH 8, and the temperature of 25 °C was more efficient in cleavage ratios ( 18 ). The present study used the incubation temperature of 25 °C for 24 h at pH 6.5 according to the IMPACT manual.…”