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Figure 4 | Microbial Cell Factories

Figure 4

From: Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

Figure 4

RP-HPLC profiles of insulin species recovered after IMAC, transpeptidation and deprotection reactions, and final purification. (A) RP-HPLC profile of the purified IP from IMAC. IP was eluted at a retention time of 19.4 minutes. (B) RP-HPLC profile of insulin species recovered after the transpeptidation reaction with i) pre-insulin H-Thr(tBu)-OtBu; ii) insulin species cleaved at B29 without the threonine ester; and iii) Insulin species cleaved at B22. (C) RP-HPLC profile of deprotection reaction in which the pre-insulin reaction mixture was incubated at room temperature for 5 minutes, with i) pre-insulin H-Thr(tBu)-OtBu; iv) insulin Thr-OtBu; and v) human insulin. (D) RP-HPLC profile of deprotection reaction in which the pre-insulin reaction mixture was incubated at room temperature for 60 minutes (E) RP-HPLC profile of purified human insulin. (F) RP-HPLC profile of mixture of human insulin and European Pharmacopoeia human insulin standard. The Phenomenex Jupiter C4 column was employed for quantification of insulin species. The identity of insulin species was determined by mass spectrometry as specified in the Materials and Methods section.

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Microbial Cell Factories

ISSN: 1475-2859

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