scholarly journals PENGARUH VARIASI KONSENTRASI METANOL DAN LAMA INDUKSI TERHADAP EKSPRESI PROINSULIN OLEH Pichia pastoris SECARA INTRASELULER

2019 ◽  
Vol 6 (1) ◽  
pp. 93
Author(s):  
Efrida Martius ◽  
Andree Triyadi ◽  
Dewi Yustika Sofia ◽  
Anis Herliyati Mahsunah
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Methanol Concentration ◽  
Diabetic Patients ◽  
Insulin Production ◽  
E Coli ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Alternative Expression ◽  
Recombinant Technology

The Effects of Variation in Methanol Concentration and Induction Time on Intracellular Proinsulin Expression by Pichia pastoris ABSTRACTDiabetes is a metabolic disorder characterized by hyperglycemia. There were 215 million diabetic patients in 2014 and the number is expected to rise in 2040. Generally, insulin is used to treat diabetic patients. Insulin production by recombinant technology has been done, though still inefficient, by using E. coli and S. cerevisiae expression system. Another alternative expression system is methylotrophic yeast Pichia pastoris. In this research, proinsulin has been expressed by P. pastoris intracellularly. P. pastoris strains used in this research were X33, GS115, and KM71H. All recombinant strains were MutS. Best cultivation media was BMGY. Proinsulin expression was observed at 25°C. Pichia pastoris strain that expressed proinsulin best was GS115-PI. It was supported by PCR in which the strain GS115-PI gave 504 bp-sized bands. Based on proinsulin formation time, the final methanol concentration of 0.5% in 72 hours was found to be the best treatment.Keywords: BMGY, methanol, phenotype, Pichia pastoris, proinsulin ABSTRAKDiabetes melitus merupakan kelainan yang ditandai dengan hiperglikemia. Penderita diabetes pada tahun 2014 di dunia mencapai 215 juta dan diperkirakan akan meningkat pada tahun 2040. Umumnya penderita diabetes diberi pengobatan insulin sehingga menunjukkan akan ada peningkatan kebutuhan insulin. Produksi insulin dengan teknologi DNA rekombinan telah dilakukan dengan menggunakan sistem ekspresi E. coli dan S. cerevisiae namun masih belum efisien. Sistem alternatif lain adalah ragi metilotropik Pichia pastoris. Dalam penelitian ini dilakukan ekspresi proinsulin dari P. pastoris secara intraseluler. Galur P. pastoris yang digunakan dalam penelitian ini adalah X33, GS115, dan KM71H. Semua galur rekombinan adalah MutS. Media tumbuh terbaik adalah BMGY. Ekspresi proinsulin terlihat pada suhu 25°C. Hasil PCR menunjukkan bahwa galur GS115-PI yang dapat menghasilkan pita amplikon berukuran 504 bp. Hasil PCR ini dibuktikan oleh hasil seleksi galur yang menunjukkan bahwa galur GS115-PI dapat mengekspresi proinsulin dibandingkan galur lainnya. Berdasarkan kecepatan pembentukan pita protein proinsulin, variasi konsentrasi akhir metanol 0,5% dengan lama induksi 72 jam merupakan perlakuan terbaik.Kata Kunci: BMGY, fenotipe, metanol, Pichia pastoris, proinsulin

scholarly journals Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation

2019 ◽  
Vol 77 (3) ◽  
Author(s):  
Rajgokul K Shanmugam ◽  
Viswanathan Ramasamy ◽  
Rahul Shukla ◽  
Upasana Arora ◽  
Sathyamangalam Swaminathan ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Surface Antigen ◽  
Zika Virus ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Mosquito Vector ◽  
Virus Envelope ◽  
Electron Microscopic ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Domain Iii

ABSTRACTZika virus (ZIKV) is an arbovirus which shares antigenic similarity and the mosquito vector with dengue viruses (DENVs). ZIKV is a neurotropic virus capable of causing congenital neurodevelopmental birth defects. As ZIKV antibodies (Abs) can potentially enhance infection by DENVs, a preventive ZIKV vaccine must be designed to eliminate antibody dependent enhancement of infection. We developed a Zika Subunit Vaccine (ZSV) consisting of two proteins, ZS and S, in a genetically pre-determined ratio of 1:4, using the methylotrophic yeast Pichia pastoris. ZS is an in-frame fusion of ZIKV envelope domain III with the Hepatitis B virus (HBV) surface antigen, and S is the un-fused HBV surface antigen. Using specific monoclonal Abs we showed the presence of ZS and S in the co-purified material which were found to co-assemble into virus-like particles (VLPs), based on dynamic light scattering and electron microscopic analyses. These VLPs were immunogenic in BALB/c mice, eliciting Abs capable of neutralizing ZIKV reporter virus particles. Further, the VLP-induced Abs did not enhance a sub-lethal DENV-2 challenge in AG129 mice. This important safety feature, coupled to the well-documented advantage of P. pastoris expression system, warrants further exploration of ZSV VLP as a possible vaccine candidate.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

2014 ◽  
Vol 1841 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

scholarly journals Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5538
Author(s):  
Zhongxuan Li ◽  
Qiang Cheng ◽  
Henan Guo ◽  
Rijun Zhang ◽  
Dayong Si
Keyword(s):  
Pichia Pastoris ◽  
High Performance ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
E Coli ◽  
Cell Membrane Permeabilization ◽  
Bactericidal Activities ◽  
The Individual ◽  
Industrial Level

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

scholarly journals Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

2013 ◽  
Vol 1831 (2) ◽  
pp. 282-290 ◽  
Author(s):  
Vasyl A. Ivashov ◽  
Karlheinz Grillitsch ◽  
Harald Koefeler ◽  
Erich Leitner ◽  
Dominic Baeumlisberger ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Lipid Droplets ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris
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