Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system

2019 ◽  
Vol 95 (3) ◽  
pp. 535-543
Author(s):  
Lingfeng Long ◽  
Yunbo Zhang ◽  
Hongyan Ren ◽  
Haiyan Sun ◽  
Fubao F Sun ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Recombinant Expression ◽  
Expression System

scholarly journals Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

1999 ◽  
Vol 22 (3) ◽  
pp. 273-283 ◽  
Author(s):  
C Sen Gupta ◽  
RR Dighe
Keyword(s):  
Pichia Pastoris ◽  
Structure Function ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Recombinant Expression ◽  
Expression System ◽  
Biologically Active ◽  
Maximal Response ◽  
Glycoprotein Hormone

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

scholarly journals Constitutive Optimized Production of Streptokinase inSaccharomyces cerevisiaeUtilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter ofPichia pastoris

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Ravi N. Vellanki ◽  
Ravichandra Potumarthi ◽  
Kiran K. Doddapaneni ◽  
Naveen Anubrolu ◽  
Lakshmi N. Mangamoori
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Expression Vector ◽  
Recombinant Expression ◽  
Expression System ◽  
Nitrogen Sources ◽  
Clot Lysis ◽  
Process Conditions ◽  
Scale Production ◽  
Activation Assay

A novel expression vector constructed from genes ofPichia pastoriswas applied for heterologous gene expression inSaccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter ofPichia pastorisinSaccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay andin vitroclot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions.

Purification of Recombinant Mouse C-Reactive Protein from Pichia Pastoris GS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography and P-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

2021 ◽  
Author(s):  
Bin Cheng ◽  
Di Wu ◽  
Ke Wu ◽  
Xiao-Ping Huang ◽  
Jian-Min Lv ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Affinity Chromatography ◽  
Protein Function ◽  
Recombinant Expression ◽  
Expression System ◽  
Post Translational Modification ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Phosphoryl Choline ◽  
Fast Flow

Abstract C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

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 rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

2006 ◽  
Vol 395 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Chiara Ciaccio ◽  
Alessandra Gambacurta ◽  
Giampiero DE Sanctis ◽  
Domenico Spagnolo ◽  
Christina Sakarikou ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Biochemical Characterization ◽  
Expression System ◽  
Gel Filtration ◽  
Proteolytic Processing ◽  
Kinetic Properties ◽  
Specific Substrate ◽  
Human Eosinophil ◽  
Eosinophil Peroxidase ◽  
Pichia Pastoris Expression System

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

scholarly journals 1P001 Construction of an expression system of canine milk lysozyme in the metylotrophic yeast Pichia pastoris

2004 ◽  
Vol 44 (supplement) ◽  
pp. S30
Author(s):  
D. Akieda ◽  
T. Aizawa ◽  
M. Yasui ◽  
Y. Nonaka ◽  
M. Watanabe ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Yeast Pichia Pastoris
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