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Journal of Multidisciplinary Applied Natural Science

e-ISSN: 2774-3047

Articles https://doi.org/10.47352/jmans.2774-3047.368

https://doi.org/10.47352/jmans.2774-3047.368

Modification of a Gene Cassette to Express Proglargine in Pichia pastoris: Elimination of Glu-Ala Repeats, Spacer and C-peptide Sequences

Uli Julia Nasution Rika Indri Astuti Aris Tri Wahyudi Dudi Hardianto Efrida Martius

Informações do autor

Uli Julia Nasution

https://orcid.org/0000-0003-1547-7883
  • ulij001@brin.go.id
  • Doctoral Program in Microbiology, Department of Biology, IPB University, Bogor-16680 (Indonesia); Research Centre for Applied Microbiology and Bureau of Organization and Human Resources, National Research and Innovation Agency, Tangerang Selatan-15311 (Indonesia)
  • Biografia não informada.

Informações do autor

Rika Indri Astuti

https://orcid.org/0000-0003-1561-6943
  • rikaindriastuti@apps.ipb.ac.id
  • Department of Biology, IPB University, Bogor-16680 (Indonesia); Biotech Center, IPB University, Bogor-16680 (Indonesia)
  • Biografia não informada.

Informações do autor

Aris Tri Wahyudi

https://orcid.org/0000-0001-7837-9557
  • ariswa@apps.ipb.ac.id
  • Department of Biology, IPB University, Bogor-16680 (Indonesia)
  • Biografia não informada.

Informações do autor

Dudi Hardianto

https://orcid.org/0000-0002-5205-1980
  • dudi003@brin.go.id
  • Research Centre for Drug and Vaccine, National Research and Innovation Agency, Tangerang Selatan-15311 (Indonesia)
  • Biografia não informada.

Informações do autor

Efrida Martius

https://orcid.org/0000-0003-4116-7897
  • efri002@brin.go.id
  • Research Centre for Drug and Vaccine, National Research and Innovation Agency, Tangerang Selatan-15311 (Indonesia)
  • Biografia não informada.
Publicado em: março 10, 2026
[1]
U. J. Nasution, R. I. Astuti, A. T. Wahyudi, D. Hardianto, e E. Martius, “Modification of a Gene Cassette to Express Proglargine in Pichia pastoris: Elimination of Glu-Ala Repeats, Spacer and C-peptide Sequences”, J. Multidiscip. Appl. Nat. Sci., mar. 2026.

Resumo

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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