Journal of Multidisciplinary Applied Natural Science
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4.8
Calculated on 05 May, 2025
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4.8
Calculated on 05 May, 2025
0.31
Powered by scimagojr.com
https://doi.org/10.47352/jmans.2774-3047.368
Uli Julia Nasution
Rika Indri Astuti
Aris Tri Wahyudi
Dudi Hardianto
Efrida Martius
The growing global prevalence of diabetes mellitus has sharply increased the demand for insulin and its analogues. Pichia pastoris is a well-established system for recombinant pro-insulin and its analogues production. However, conventional gene constructs often include additional sequences, such as Glu-Ala repeats, spacer peptides, and c-peptides that complicate downstream processing and reduce efficiency. This study aimed to construct and express a proglargine (PG) gene cassette lacking the Glu-Ala repeats, spacer, and c-peptide in P. pastoris GS115 to obtain a uniform PG protein. The recombinant vector propagated in Escherichia coli TOP10F’, then expressed in P. pastorisGS115. Selected transformants were cultivated in YPG medium, then induced with 1% and 2% methanol daily in BMMY. The optimum methanol concentration further evaluated in ½ BSM induction medium. The result demonstrated that optimal PG expression was achieved with 2% methanol induction in BMMY, producing higher levels than those with ½ BSM. Among the transformants, PG.c2 produced the highest PG protein in BMMY medium induced with 2% methanol. Dot-blot analysis confirmed the secretion of PG, and LC-HRMS analysis demonstrated 100% amino acid sequence coverage of PG, confirming the integrity and completeness of the expressed protein. This study presented a newly modified PG gene cassette, inserted into pPICZαA vector, to express uniform secreted PG in P. pastoris GS115. By simplifying the precursor structure, a more homogeneous precursor product can be obtained, which is expected to simplify purification and also the downstream enzymatic process of PG into mature insulin glargine.
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