In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.
Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.
Production of sufficient insulin at a more affordable price is necessary. The increase in the number of people living with diabetes puts more burden on healthcare and the economy. P. pastoris is a promising host to produce human insulin precursors at a high yield in minimal medium and secretes low levels of endogenous protein impurities. Production of the precursor involves several parameters, including glycerol concentration, culture density, methanol concentration, and medium composition. This study evaluated the effect of those parameters on protein expression in the flask culture. Subsequently, fermentation in the bioreactor was carried on according to the information obtained from flask culture. Methanol feeding to induce protein expression was undertaken by pulses and fed-batch modes. The fed-batch method was modified from a standard technique by incorporating constant flow rates with variable feed concentrations. Cell density was determined based on optical density measurement at 600 nm and dry cell weight. Tricine SDS-PAGE and reversed-phase HPLC conducted protein analysis. The pulse feeding produced higher precursor concentrations at ~445 mg/L than modified fed-batch feeding at ~267 mg/L. However, the modified fed-batch feeding can be an alternative to producing human insulin precursors when a standard fed-batch feeding with variable flow rates and 100% (v/v) methanol feed is difficult to apply.
The methylotrophic yeast, Pichia pastoris, is one of the preferred yeast hosts for recombinant protein expression. It has been developed as a potential host to express a high level of recombinant proteins, and to achieve efficient secretion as well as growth to very high cell densities. Previously, we have obtained 19 P. pastoris recombinant clones harboring synthetic insulin precursor (IP) expression cassette integrated into their genomes through homologous recombination. To select P. pastoris recombinant clones which exhibit high levels of protein expression, we conducted secreted expressions of IP protein in shake flasks. The secretion of IP into the culture supernatants was verified by SDS-PAGE. IP protein concentrations were estimated using ImageJ by applying lysozyme as standard. All of the 19 P. pastoris recombinant clones were confirmed to secrete the IP protein into their culture supernatants, and a single protein band with a molecular size of approximately 7 kDa was found in the SDS-PAGE gel. The six highest IP-expressing clones were selected for second screening in shake flasks. We selected three recombinant clones (CL-3, CL-4, and CL-18), which secreted the highest levels of IP proteins compared to the other clones. The secreted IP concentrations estimated by ImageJ for clones CL-3, CL-4, and CL-18 were 1230, 1143, and 1010 mg/L, respectively.
<p>Kota Surakarta meraih peringkat ke-2 Kota Layak Huni di Indonesia dengan nilai rata-rata 69,38% dari nilai rata-rata nasional berdasarkan survey Ikatan Ahli Perencanaan (IAP) pada tahun 2014 melalui Most Index Livable City. Salah satu aspek yang dinilai adalah aspek transportasi. Kota Surakarta memiliki letak strategis yang menghubungakan kota-kota besar di Pulau Jawa sehingga kondisi tersebut mempengaruhi pertumbuhan ekonomi di Surakarta yang berdampak pada sistem dan pola transportasi. Tujuan dari penelitian ini yaitu, untuk mengetahui kesesuaian sistem transportasi umum di Kota Surakarta terhadap konsep Transportation for Livable City. Penelitian ini menggunakan metode kuantitatif dengan analisis skoring skala Guttman. Analisis skoring dilakukan pada tiap parameter. Dari hasil analisis menunjukkan bahwa sistem transportasi di Kota Surakarta termasuk ke dalam kategori mendekati tidak sesuai. Hal ini diketahui dari adanya beberapa variabel yang tidak sesuai dengan kriteria konsep transportation for livable city, yaitu jalur sepeda, jalur pedestrian, titik transit, dan jalur angkutan umum. Hasil akhir yang diperoleh menyatakan bahwa varibel-variabel tersebut mengalami penurunan kualitas yang menyebabkan variabel tersebut tidak sesuai dengan konsep transportation for livable city.</p>
Current recombinant human insulin production utilizes two major expression systems, Escherichia coli and Saccharomyces cerevisiae-based expression systems. Methylotropic yeast, Pichia pastoris, has appeared as a promising alternate yeast recombinant host for insulin precursor (IP) expression due to its ability to produce high titers, efficient secretion, and growth to very high cell densities. Similar to the S. cerevisiae system, P. pastoris secreted soluble IP into the culture supernatant. In the previous study, we have established P. pastoris recombinant clones harboring synthetic insulin precursor (IP) expression cassette integrated into their genomes through homologous recombination. It is essential to verify that the expression cassettes of the IP encoding gene remain stably integrated with the genome during such prolonged methanol induction. Therefore, we aimed to analyze the stability of one recombinant clone (CL-4) expressing the human insulin precursor by verifying the stable integration of the IP expression cassette into the genome by PCR, and the IP protein expression after prolonged methanol induction over 70 generations. We found that the expression cassette was stably integrated into the genome of the CL-4 recombinant clone and the IP expression was sustained after 72 generations of cultivations in the culture and induction media without antibiotic selection.
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