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 Cloning of PpKAR2 and PpPDI1 genes promoters from yeast Pichia pastoris, an evaluation of their activity and efficacy for the heterologous genes expression

2018 ◽  
Vol 16 (2) ◽  
pp. 50-59 ◽  
Author(s):  
Mikhail A. Tsygankov ◽  
Marina V. Padkina
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Human Interferon ◽  
Heterologous Proteins ◽  
Reporter System ◽  
Promoter Regions ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Pichia Pastoris ◽  
Yeast Genes

Background. Yeast Pichia pastoris is successfully used in biotechnology, with their help synthesized various compounds. Promoters are a key factor in the productivity of an expression system, since they determine the expression level of a heterologous gene. The aim of our work was to study the promoter regions of the PpKAR2 and PpPDI1 genes and to evaluate their use for effective expression of heterologous genes. Materials and Methods. To evaluate the activity of promoters, we used a reporter system based on the structural gene of acid phosphatase of yeast Saccharomyces cerevisiae – PHO5. To determine the effect of overproduction of native and heterologous protein on the activity of the promoters under study, we used the producer strains of P. pastoris protein disulfide isomerase and maize delta-zein. To evaluate the effectiveness of the use of the promoters under study for the expression of heterologous genes, we have expressed under their control a gene encoding human interferon-alpha16. Results. The promoters of the yeast genes – PpKAR2 and PpPDI1 were cloned. Their activity was compared with the promoter of the PpAOX1 gene in the native strains, as well as in strains with overproduction of native and heterologous proteins. Under the control of these promoters, the gene encoding human interferon-alpha 16 is expressed. Conclusion. The promoters studied were weaker than the promoter of the AOX1 gene, but increase their activity in response to the production of heterologous proteins and can be used to express hete­rologous genes.

Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain

2011 ◽  
Vol 47 (3) ◽  
pp. 279-287 ◽  
Author(s):  
A. M. Rozhkova ◽  
A. S. Sereda ◽  
N. V. Tsurikova ◽  
A. K. Nurtaeva ◽  
M. V. Semenova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Recombinant Strain ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Heterologous Gene ◽  
Aspergillus Awamori ◽  
Gene Expression System

scholarly journals Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon

2005 ◽  
Vol 86 (4) ◽  
pp. 1045-1053 ◽  
Author(s):  
Rainer Gehrke ◽  
Franz X. Heinz ◽  
Nancy L. Davis ◽  
Christian W. Mandl
Keyword(s):  
Gene Expression ◽  
Translational Control ◽  
Fluorescent Protein ◽  
Heterologous Gene Expression ◽  
Surface Proteins ◽  
Heterologous Gene ◽  
Expression Level ◽  
Vector System ◽  
Egfp Expression ◽  
Foreign Gene

The flavivirus tick-borne encephaltis virus (TBEV) was established as a vector system for heterologous gene expression. The variable region of the genomic 3′ non-coding region was replaced by an expression cassette consisting of the reporter gene enhanced green fluorescent protein (EGFP) under the translational control of an internal ribosomal entry site element, both in the context of an infectious virus genome and of a replicon lacking the genes of the surface proteins prM/M and E. The expression level and the stability of expression were measured by fluorescence-activated cell-sorting analysis and compared to an established alphavirus replicon vector derived from Venezuelan equine encephaltis virus (VEEV), expressing EGFP under the control of its natural subgenomic promoter. On the first day, the alphavirus replicon exhibited an approximately 180-fold higher expression level than the flavivirus replicon, but this difference decreased to about 20- and 10-fold on days 2 and 3, respectively. Four to six days post-transfection, foreign gene expression by the VEEV replicon vanished almost completely, due to extensive cell killing. In contrast, in the case of the TBEV replicon, the percentage of positive cells and the amount of EGFP expression exhibited only a moderate decline over a time period of almost 4 weeks. The infectious TBEV vector expressed less EGFP than the TBEV replicon at all times. Significant expression from the infectious vector was maintained for four cell-culture passages. The results indicate that the VEEV vector is superior with respect to achieving high expression levels, but the TBEV system may be advantageous for applications that require a moderate, but more enduring, gene expression.

scholarly journals Bestowing Inducibility on the Cloned Methanol Dehydrogenase Promoter (PmxaF) of Methylobacterium extorquens by Applying Regulatory Elements of Pseudomonas putida F1

2006 ◽  
Vol 72 (12) ◽  
pp. 7723-7729 ◽  
Author(s):  
Young J. Choi ◽  
Lyne Morel ◽  
Denis Bourque ◽  
Alaka Mullick ◽  
Bernard Massie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Putida ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Regulatory Elements ◽  
Heterologous Gene ◽  
Expression Vectors ◽  
Transcriptional Level ◽  
Broad Host Range ◽  
Methylobacterium Extorquens

ABSTRACT P mxaF is a strong methanol-inducible promoter in Methylobacterium extorquens. When this promoter is cloned in expression vectors and used to drive heterologous gene expression, methanol inducibility is either greatly reduced or entirely lost. In order to bestow inducibility upon the cloned P mxaF promoter in expression vectors, we adopted combinational methods (regulatory elements of the Pseudomonas putida F1 cym and cmt operons and Tn7 transposon system) to control reporter gene expression at the transcriptional level in M. extorquens. An operator fragment (26 nucleotides) of the cmt operon was inserted downstream of the cloned P mxaF promoter in the broad-host-range expression vector (pCHOI3). The repressor gene (cymR) located upstream of the cym operon in P. putida F1 was amplified by PCR. To avoid cellular toxicity for M. extorquens caused by the overexpression of CymR, single and/or double copies of cymR were integrated into the chromosome of M. extorquens using the mini-Tn7 transposon system. Cultures containing the chromosomally integrated cymR gene were subsequently transformed with pCHOI3 containing modified P mxaF (i.e., P mxaF plus operator). In this construct, inducibility is afforded by cumate (p-isopropylbenzoate). In this report, we describe the inducible and tightly regulated expression of heterologous genes (bgl [for β-galactosidase], est [for esterase], and gfp [for green fluorescent protein]) in M. extorquens. This is the first documented example of an inducible/regulated heterologous gene expression system in M. extorquens.

scholarly journals A Derepression System Based on the Bacillus subtilis Sporulation Pathway Offers Dynamic Control of Heterologous Gene Expression

2007 ◽  
Vol 73 (7) ◽  
pp. 2390-2393 ◽  
Author(s):  
Reindert Nijland ◽  
Jan-Willem Veening ◽  
Oscar P. Kuipers
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Dynamic Control ◽  
Inducible Promoter ◽  
Heterologous Gene ◽  
Gene Regulatory

ABSTRACT By rewiring the sporulation gene-regulatory network of Bacillus subtilis, we generated a novel expression system relying on derepression. The gene of interest is placed under the control of the abrB promoter, which is active only when Spo0A is absent, and Spo0A is controlled via an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter.

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