Enhancement in Human Insulin Precursor Secretion by Pichia pastoris through Modification of Expression Conditions

Nurdiani, Dini; Hariyatun, Hariyatun; Utami, Ning Wikan; Putro, Eko Wahyu; Kusharyoto, Wien

doi:10.4308/hjb.29.1.22-30

Cited by 6 publications

(3 citation statements)

References 22 publications

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“…Another finding is that the Mut + strains are sensitive to transient high methanol concentrations [ 53 – 56 ]. On the other hand, in our previous study, CL4 has an optimal methanol induction for the HIP expression of about 2–4% [ 57 ].…”

Section: Discussionmentioning

confidence: 92%

Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

Utami¹,

Nurdiani²,

Hariyatun³

et al. 2023

Journal of Genetic Engineering and Biotechnology

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Background Human insulin was the first FDA-approved biopharmaceutical drug produced through recombinant DNA technology. The previous studies successfully expressed recombinant human insulin precursors (HIP) in Pichia pastoris truncated and full-length α-factor recombinant clones. The matting α-factor (Matα), a signal secretion, direct the HIP protein into the culture media. This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM). Results ImageJ analysis of the HIP’s SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones. Conclusions CL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. This research confirmed that deletion of some regions of the secretory signal sequence significantly improved the efficiency of HIP protein expression in P. pastoris.

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Section: Discussionmentioning

confidence: 92%

Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

Utami¹,

Nurdiani²,

Hariyatun³

et al. 2023

Journal of Genetic Engineering and Biotechnology

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“…For example, mutants of the α-MF-where at position 42, amino acid residue Leu is replaced by Ser, and at position 83, Asp is substituted with Glu-increase the productivity of the recombinant protein by almost threefold as compared with the wild type [52]. Deletions in the α-MF pro-region (∆57-70) have improved the efficiency of expression of some heterologous genes in K. phaffii cells by almost an order of magnitude [100][101][102].…”

Section: The Choice Of a Signal Sequencementioning

confidence: 99%

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Khlebodarova,

Bogacheva,

Zadorozhny

et al. 2024

Microorganisms

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In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker’s yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species’ several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

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“…The recombinant strain was engineered and selected previously [4,9]. A selection media, Yeast Peptone Dextrose (YPD) agar with zeocin 100 µg/mL, was used to regenerate the employed colony [10,11]. Media of half concentration basal salt media (½ BSM) was used as the preculture and bioreactor media [12].…”

Section: Strains and Mediamentioning

confidence: 99%

Expression of recombinant human insulin precursor by Pichia pastoris in a 10 liter bioreactor

Puspitasari,

Mahsunah,

Nurdiani

et al. 2023

IOP Conf. Ser.: Earth Environ. Sci.

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Recombinant insulin is a vital medicine for diabetic patients. This hormone is produced by microbes such as Pichia pastoris that carry the recombinant gene of a human insulin precursor (HIP). Large-scale protein production involves a bioreactor to promote the optimal condition for the yeast to express the protein target. In order to obtain a large amount of insulin, the cultivation of recombinant P.pastoris/pD902-IP carrying human insulin precursor gene in a bioreactor 10 Liter was developed. The isolate was cultivated in a half concentration of basal salt media for 124.5 hours. Induction of the protein was done by continual methanol feeding. The fermentation condition was set to have a temperature at 28°C, agitation at 300 rpm, aeration at 2 L/min and a pH value of around 5. Dry cell weight (DCW) was measured, and HPLC quantified the content of HIP, glycerol and methanol. This work’s DCW and HIP concentrations were 46.5 g/L and 928 mg/L, respectively. The results can be higher by increasing the number of cells in the culture or extending the cultivation time so that the HIP concentration may exceed 1 g/L.

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Enhancement in Human Insulin Precursor Secretion by Pichia pastoris through Modification of Expression Conditions

Cited by 6 publications

References 22 publications

Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Expression of recombinant human insulin precursor by Pichia pastoris in a 10 liter bioreactor

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