The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris

Scorer, Carol A.; Buckholz, Richard G.; Clare, Jeffrey J.; Romanos, Michael A.

doi:10.1016/0378-1119(93)90454-b

Cited by 151 publications

(83 citation statements)

References 32 publications

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“…This was presumed to be due to premature termination of transcripts in AT-rich regions and has been observed in Pichia and other eukaryotic systems. In particular, the sequence TTTTTATA is known to promote premature polyadenylation in Saccharomyces cerevisiae, and a similar sequence has been shown to cause termination when the human immunodeficiency virus type 1 gp120 protein is expressed in P. pastoris (25). When the sequence TTTTTATTC present in rat M-CPT I (encoding amino acids 122-124) was mutated (without changing the amino acid sequence; see "Experimental Procedures"), higher levels of expressed activity were achieved, although it was still necessary to select for high copy number integrants.…”

Section: Expression Of Wild-type Rat L-and M-cpt Imentioning

confidence: 99%

Use of Six Chimeric Proteins to Investigate the Role of Intramolecular Interactions in Determining the Kinetics of Carnitine Palmitoyltransferase I Isoforms

Jackson

Cameron

Fraser

et al. 2000

Journal of Biological Chemistry

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Section: Expression Of Wild-type Rat L-and M-cpt Imentioning

confidence: 99%

Use of Six Chimeric Proteins to Investigate the Role of Intramolecular Interactions in Determining the Kinetics of Carnitine Palmitoyltransferase I Isoforms

Jackson

Cameron

Fraser

et al. 2000

Journal of Biological Chemistry

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“…The construct was used to transform P. pastoris strain X-33. After induction, the use of S. cerevisiae alphafactor prepro-leader sequence will target the protein to the secretory pathway and finally secreted into the medium for simple analysis [24][25]. In this study, information on optimal concentration of methanol induction as well as variation incubation times is presented.…”

Section: Resultsmentioning

confidence: 99%

EFFECT OF METHANOL INDUCTION AND INCUBATION TIME ON EXPRESSION OF HUMAN ERYTHROPOIETIN IN METHYLOTROPIC YEAST Pichia pastoris

Santoso¹,

Herawati²,

Rubiana³

2012

MST

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Erythropoietin (EPO) is a glycoprotein hormone consists of 165 amino acids and has molecular mass of 30,400 Daltons. The large quantities of these hormone required to satisfy clinical demand are currently met by recombinant expression in mammalian cell, namely chinese hamster ovary (CHO). Pichia pastoris has become popular yeast based protein production systems to substitute mammalian expression systems. P. pastoris is capable to use methanol as sole carbon and energy source. In this study, recombinant human EPO (rhEPO) protein obtained by expressing the hEPO gene in methylotropic yeast P. pastoris, strain X33. The present work was carried out to study the optimal methanol concentration for induction and the incubation time to obtain rhEPO protein. To perform this study, the transformed P. pastoris was induced with various concentrations of methanol (0%, 0.5%, 1%, 2.5%, 5%, 10%, and 20%) and incubation times (0 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours). The results demonstrate that the highest protein expression level occurred at concentration of 2.5% methanol induction, while the optimal incubation time was at 48 hrs. Abstrak Pengaruh Induksi Methanol dan Waktu Inkubasi terhadap Ekspresi Eritropoietin Manusia pada KhamirMetilotropik Pichia pastoris. Eritropoietin (EPO) adalah hormon glikoprotein yang terdiri dari 165 asam amino dan memiliki berat molekul sebesar 30.400 Daltons. Sebagian besar kebutuhan EPO didapatkan dari hasil sintesis pada sel mamalia Chinese hamster ovary (CHO). Pichia pastoris adalah sejenis khamir yang populer digunakan untuk menggantikan sistem ekspresi pada sel mamalia. P. pastoris dapat menggunakan metanol sebagai satu-satunya sumber energi karbon. Pada studi ini, protein rekombinan EPO (rhEPO) disintesis dengan cara mengekspresikan gen hEPO pada khamir metilotropik P. pastoris strain X33. Studi ini dilakukan untuk mengetahui konsentrasi metanol dan waktu inkubasi yang optimal untuk mensintesis rhEPO. Pada studi ini konsentrasi metanol yang digunakan adalah 0%, 0.5%, 1%, 2.5%, 5%, 10%, dan 20%. Sedangkan waktu inkubasi yang digunakan adalah 0 jam, 24 jam, 48 jam, 72 jam, 96 jam, 120 jam, and 144 jam. Hasil penelitian ini menunjukkan bahwa ekspresi protein yang tertinggi terjadi pada konsentrasi metanol sebesar 2.5% dan waktu inkubasi selama 48 jam.

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“…The native N-terminal signal of ZmCKX1 was replaced with the ␣-factor propeptide of Saccharomyces cerevisiae to ensure efficient secretion of expressed ZmCKX1 peptides from Pi. pastoris (22).…”

Section: Scaffold-peptide Construction For Expression In Pi Pastorismentioning

confidence: 99%

Combinatorially selected defense peptides protect plant roots from pathogen infection

Fang

Laskey²,

Huang³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Agricultural productivity and sustainability are continually challenged by emerging and indigenous pathogens. Currently, many pathogens can be combated only with biocides or environmentally dangerous fumigants. Here, we report a rapid and pathogenspecific strategy to reduce infection by organisms that target plant roots. Combinatorially selected defense peptides, previously shown to effect premature encystment of Phytophthora capsici zoospores, were fused to maize cytokinin oxidase͞dehydrogenase as a display scaffold. When expressed in tomato roots, the peptidescaffold constructs were secreted and accumulated to sufficient concentrations in the rhizosphere to induce zoospore encystment and thereby deter taxis to the root surface. Pathogen infection was significantly inhibited in roots expressing bioactive peptides fused to the maize cytokinin oxidase͞dehydrogenase scaffold. This peptide-delivery technology is broadly applicable for rapid development of plant defense attributes against plant pathogens.disease resistance ͉ Phytophthora

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The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris

Cited by 151 publications

References 32 publications

Use of Six Chimeric Proteins to Investigate the Role of Intramolecular Interactions in Determining the Kinetics of Carnitine Palmitoyltransferase I Isoforms

Use of Six Chimeric Proteins to Investigate the Role of Intramolecular Interactions in Determining the Kinetics of Carnitine Palmitoyltransferase I Isoforms

EFFECT OF METHANOL INDUCTION AND INCUBATION TIME ON EXPRESSION OF HUMAN ERYTHROPOIETIN IN METHYLOTROPIC YEAST Pichia pastoris

Combinatorially selected defense peptides protect plant roots from pathogen infection

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