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ABSTRACT: The human secretory leukocyte protease inhibitor (SLPI) is an 11.7 kD cysteine-rich protein that has been shown to possess anti-protease, anti-inflammatory, and antimicrobial properties. By using a Pichia pastoris strain that overproduces protein disulfide isomerase (PDI), we obtained greater than fivefold higher levels of SLPI than in strains expressing normal levels of PDI and containing multiple copies of the SLPI gene. Elevated levels of PDI also enhanced the specific activity of the secreted SLPI by helping it achieve a proper tertiary structure. Mass spectrometry analysis indicated a greater number of disulfide bonds in the SLPI produced by the PDI overexpression strain compared to the SLPI produced in strains with normal PDI levels. Although others have utilized a similar strategy to increase yield, we believe that this is the first example of PDI overexpression being demonstrated to enhance the folding and thus increase the biological activity of a protein produced in the yeast P. pastoris.
Biochemical and Biophysical Research Communications 10/2010; 402(3):519-24. · 2.48 Impact Factor
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ABSTRACT: The cardiovascular effects of estrogen are mediated in part by augmenting the function of endothelial nitric oxide synthase. Endothelial nitric oxide synthase activity is dependent on many cofactors including Ca(2+). Hence, we investigated the effect of chronic 17 beta-estradiol treatment on the intracellular Ca(2+) concentration and endothelial nitric oxide synthase protein expression in the human endothelial cell line, EA.hy926, using spectrofluorometry and Western blot, respectively. Inhibiting the sarco(endo)plasmic reticulum Ca(2+) ATPase with thapsigargin caused an increase in the intracellular Ca(2+) concentration, which was higher in chronically 17 beta-estradiol-treated (1muM, 24h) cells loaded with Fura-2-acetoxymethyl ester compared to vehicle-treated cells, suggesting a higher endoplasmic reticulum Ca(2+) content in 17 beta-estradiol-treated cells. An enhanced Ca(2+) influx pathway in chronically 17 beta-estradiol-treated cells was also observed. In addition, 17 beta-estradiol-treated cells expressed higher levels of endothelial nitric oxide synthase protein in comparison to vehicle-treated cells. The chronic effect of 17 beta-estradiol on Ca(2+) homeostasis and endothelial nitric oxide synthase expression was attenuated with the nonselective estrogen receptor inhibitor, ICI 182,780 (10muM, 7alpha, 17beta-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl] estra-1,3,5(10)-triene-3,17-diol). Furthermore, analysis of the thapsigargin-evoked Ca(2+) response in chronically 17 beta-estradiol-treated estrogen receptor alpha-knockdown cells showed no significant difference in Ca(2+) response compared to vehicle-treated estrogen receptor alpha-knockdown cells, indicating that the regulation of Ca(2+) homeostasis by 17 beta-estradiol is mediated through an estrogen receptor alpha-dependent pathway. These data revealed an estrogen receptor alpha-dependent modulation of Ca(2+) homeostasis accompanying the enhancement of endothelial nitric oxide synthase expression in 17 beta-estradiol-treated human endothelial cells.
European journal of pharmacology 03/2010; 630(1-3):92-9. · 2.59 Impact Factor
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Zhiguo Li,
Wilson Leung,
Amy Yon,
John Nguyen,
Vincent C Perez,
Jane Vu,
William Giang,
Linda T Luong,
Tracy Phan,
Kate A Salazar,
Seth R Gomez,
Colin Au,
Fan Xiang,
David W Thomas,
Andreas H Franz,
Joan Lin-Cereghino, Geoff P Lin-Cereghino
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ABSTRACT: The Escherichia coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins.
Protein Expression and Purification 03/2010; 72(1):113-24. · 1.59 Impact Factor
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Zhiguo Li,
Allison Moy,
Kirti Sohal,
Carolyn Dam,
Peter Kuo,
James Whittaker,
Mei Whittaker,
Nejat Düzgünes,
Krystyna Konopka,
Andreas H Franz,
Joan Lin-Cereghino, Geoff P Lin-Cereghino
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ABSTRACT: The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.
Protein Expression and Purification 07/2009; 67(2):175-81. · 1.59 Impact Factor
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ABSTRACT: The enhanced green fluorescent protein (EGFP) is a macromolecule which fluoresces green under specific wavelengths of light. It has been widely used as a tool to study cell structure and function. EGFP-PTS1 is a fusion of the EGFP gene and a peroxisome targeting sequence (PTS1). Attachment of the PTS1 localizes EGFP to the peroxisome, an organelle found in eukaryotic cells. In this work, DNA encoding an EGFP-PTS1 fusion was inserted into a plasmid which contained a selectable marker gene for adenine biosynthesis (ADE1). The plasmid, called pCO1, was transformed into an adenine auxotrophic strain (ade1) of the yeast Pichia pastoris. The ade1 strain, which is pink in color, could not synthesize its own adenine and thus could not survive without this nitrogenous base in its growth medium. The ADE1 gene on pCO1 acted as a form of selection to identify cells transformed by the plasmid. Colonies transformed by pCO1 were white in color and could grow in medium without the supplemental addition of adenine. Cells harboring pCO1 expressed EGFP-PTS1 protein when grown on methanol because the gene fusion was put under the control of the methanol-inducible AOX1 promoter. By utilizing fluorescence microscopy, we show that these pCO1-transformed yeast express EGFP-PTS1 and localize it to their peroxisomes. The experimental method detailed here has been modified to teach concepts of yeast genetics and protein expression to large numbers of laboratory students in an undergraduate setting.
BIOS journal 01/2009;
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Joan Lin-Cereghino,
Matthew D Hashimoto,
Allison Moy,
James Castelo,
Claire C Orazem,
Peter Kuo,
See Xiong,
Vishal Gandhi,
Christopher T Hatae,
Alex Chan, Geoff P Lin-Cereghino
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ABSTRACT: The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan(r) gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANalpha B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.
Yeast 05/2008; 25(4):293-9. · 1.89 Impact Factor
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ABSTRACT: Selection of both an appropriate expression vector and corresponding strain is crucial for successful expression of heterologous
proteins in Pichia pastoris. This chapter explores both the standard and new vector/strain options available for protein expression in this yeast. Incorporated
into expression vectors are selectable markers based on biosynthetic pathway genes, dominant drug resistance, or the P. pastoris formaldehyde dehydrogenase gene (FLD1). Novel strains available for expression include those that increase secretion of heterologous protein by overexpressing
eukaryotic protein disulfide isomerase, and those that decrease hyperglycosylation or provide human-type glycosylation. This
chapter also discusses methods to create multicopy strains that will potentially provide optimized expression of recombinant
proteins in P. pastoris.
Key WordsExpression vectors–plasmids–expression strains–heterologous protein expression in Pichia pastoris
02/2008: pages 11-25;
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ABSTRACT: Selection of both an appropriate expression vector and corresponding strain is crucial for successful expression of heterologous proteins in Pichia pastoris. This chapter explores both the standard and new vector/strain options available for protein expression in this yeast. Incorporated into expression vectors are selectable markers based on biosynthetic pathway genes, dominant drug resistance, or the P. pastoris formaldehyde dehydrogenase gene (FLD1). Novel strains available for expression include those that increase secretion of heterologous protein by overexpressing eukaryotic protein disulfide isomerase, and those that decrease hyperglycosylation or provide human-type glycosylation. This chapter also discusses methods to create multicopy strains that will potentially provide optimized expression of recombinant proteins in P. pastoris.
Methods in molecular biology (Clifton, N.J.) 02/2007; 389:11-26.
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Microscopy today 10/2006; 14(5):36-37.
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ABSTRACT: We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.
FEMS Yeast Research 08/2005; 5(10):935-42. · 2.40 Impact Factor
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BioTechniques 02/2005; 38(1):44, 46, 48. · 2.67 Impact Factor
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ABSTRACT: The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels. Using this system, recent advances have been made in the quality of recombinant proteins in fermenter culture and in the quality of the protein product, namely improved secretion signals and glycosylation patterns.
Current Opinion in Biotechnology 09/2002; 13(4):329-32. · 7.71 Impact Factor
