scholarly journals Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis

2009 ◽  
Vol 75 (9) ◽  
pp. 2792-2797 ◽  
Author(s):  
Abul Kalam Azad ◽  
Yoshihiro Sawa ◽  
Takahiro Ishikawa ◽  
Hitoshi Shibata
Keyword(s):  
Plasma Membrane ◽  
Pichia Pastoris ◽  
Heterologous Expression ◽  
Osmotic Shock ◽  
Expression System ◽  
Water Channel ◽  
Gating Mechanism ◽  
Growth Assay ◽  
Multicellular Organisms

ABSTRACT Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

scholarly journals In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris

2006 ◽  
Vol 72 (2) ◽  
pp. 1507-1514 ◽  
Author(s):  
Mark J. Daniels ◽  
Malcolm R. Wood ◽  
Mark Yeager
Keyword(s):  
Pichia Pastoris ◽  
Linear Relationship ◽  
Osmotic Shock ◽  
Water Channel ◽  
Yeast Cells ◽  
Mercury Chloride ◽  
Functional Assay ◽  
Channel Activity ◽  
Wild Type

ABSTRACT The water channel protein PvTIP3;1 (α-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.

scholarly journals Expression and Purification of C-Peptide Containing Insulin UsingPichia pastorisExpression System

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Mohammed N. Baeshen ◽  
Thamer A. F. Bouback ◽  
Mubarak A. Alzubaidi ◽  
Roop S. Bora ◽  
Mohammed A. T. Alotaibi ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Restriction Analysis ◽  
Expression System ◽  
Global Market ◽  
Biologically Active ◽  
Dependent Diabetes Mellitus ◽  
Recombinant Human Insulin ◽  
C Peptide ◽  
Recombinant Insulin

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeastPichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed intoPichia pastoris SuperMan5strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA inPichiagenome was confirmed by PCR using insulin specific primers. Expression of insulin inPichiaclones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level usingPichia pastorisexpression system.

scholarly journals Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaowen Sun ◽  
Hui Liu ◽  
Peng Wang ◽  
Li wang ◽  
Wenfeng Ni ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Heterologous Expression ◽  
Biosynthetic Pathway ◽  
Initial Conditions ◽  
Expression System ◽  
Value Added ◽  
Theoretical Research ◽  
High Yield ◽  
Yield Strain ◽  
Synthetic Pathway

Abstract Background With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. Results Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. Conclusions In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.

scholarly journals Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

2014 ◽  
Vol 204 (4) ◽  
pp. 559-573 ◽  
Author(s):  
Alex J. Smith ◽  
Byung-Ju Jin ◽  
Julien Ratelade ◽  
Alan S. Verkman
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Water Channel ◽  
Aquaporin 4 ◽  
Cellular Functions ◽  
Aggregation State ◽  
Functional Roles ◽  
State Dependent ◽  
And Function

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.

scholarly journals Expression and characterisation of fully posttranslationally modified cellular prion protein in Pichia pastoris

2013 ◽  
Vol 394 (11) ◽  
pp. 1475-1483
Author(s):  
Jendrik Marbach ◽  
Peter Zentis ◽  
Philipp Ellinger ◽  
Henrik Müller ◽  
Eva Birkmann
Keyword(s):  
Plasma Membrane ◽  
Pichia Pastoris ◽  
Prion Protein ◽  
Prion Diseases ◽  
Phosphatidyl Inositol ◽  
Cellular Prion Protein ◽  
Glycosylation Pattern ◽  
Signal Sequences

Abstract Prion diseases are fatal neurodegenerative diseases which occur as sporadic, genetic, and transmissible disorders. A molecular hallmark of prion diseases is the conformational conversion of the host-encoded cellular form of the prion protein (PrPC) into its misfolded pathogenic isoform (PrPSc). PrPSc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrPC to PrPSc is a major field in prion research. PrPC is glycosylated and attached to the plasma membrane via its glycosyl phosphatidyl inositol (GPI)-anchor. In this study we established and characterised the expression of fully posttranslationally modified mammalian Syrian golden hamster PrPC in the yeast Pichia pastoris using native PrPC-specific N- and C-terminal signal sequences. In vivo as well as in vitro-studies demonstrated that the signal sequences controlled posttranslational processing and trafficking of native PrPC, resulting in PrPC localised in the plasma membrane of P. pastoris. In addition, the glycosylation pattern of native PrPC could be confirmed.

The Aquaporin TcAQP1 of the Desert Truffle Terfezia claveryi Is a Membrane Pore for Water and CO2 Transport

2012 ◽  
Vol 25 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Alfonso Navarro-Ródenas ◽  
Juan Manuel Ruíz-Lozano ◽  
Ralf Kaldenhoff ◽  
Asunción Morte
Keyword(s):  
Heterologous Expression ◽  
Mycorrhizal Fungi ◽  
Expression System ◽  
Expression Patterns ◽  
Mycorrhizal Fungus ◽  
Water Channel ◽  
Water Conductivity ◽  
Membrane Pore ◽  
Aquaporin Gene ◽  
Terfezia Claveryi

Terfezia claveryi is a hypogeous mycorrhizal fungus belonging to the so-called “desert truffles,” with a good record as an edible fungus and of considerable economic importance. T. claveryi improves the tolerance to water stress of the host plant Helianthemum almeriense, for which, in field conditions, symbiosis with T. claveryi is valuable for its survival. We have characterized cDNAs from T. claveryi and identified a sequence related to the aquaporin gene family. The full-length sequence was obtained by rapid amplification of cDNA ends and was named TcAQP1. This aquaporin gene encoded a functional water-channel protein, as demonstrated by heterologous expression assays in Saccharomyces cerevisiae. The mycorrhizal fungal aquaporin increased both water and CO2 conductivity in the heterologous expression system. The expression patterns of the TcAQP1 gene in mycelium, under different water potentials, and in mycorrhizal plants are discussed. The high levels of water conductivity of TcAQP1 could be related to the adaptation of this mycorrhizal fungus to semiarid areas. The CO2 permeability of TcAQP1 could be involved in the regulation of T. claveryi growth during presymbiotic phases, making it a good candidate to be considered a novel molecular signaling channel in mycorrhizal fungi.

scholarly journals The Variable Domain of a Plant Calcium-dependent Protein Kinase (CDPK) Confers Subcellular Localization and Substrate Recognition for NADPH Oxidase

2013 ◽  
Vol 288 (20) ◽  
pp. 14332-14340 ◽  
Author(s):  
Shuta Asai ◽  
Tatsushi Ichikawa ◽  
Hironari Nomura ◽  
Michie Kobayashi ◽  
Yusuke Kamiyoshihara ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Subcellular Localization ◽  
Expression System ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Golgi Network ◽  
Respiratory Burst Oxidase

Calcium-dependent protein kinases (CDPKs) are Ca2+ sensors that regulate diverse biological processes in plants and apicomplexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.

scholarly journals Heterologous Expression of a Membrane-Spanning Auxin Importer: Implications for Functional Analyses of Auxin Transporters

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
David John Carrier ◽  
Norliza Tendot Abu Bakar ◽  
Karen Lawler ◽  
James Matthew Dorrian ◽  
Ameena Haider ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Functional Assay ◽  
Expression Systems ◽  
Heterologous Expression Systems ◽  
Membrane Spanning ◽  
High Level

Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

scholarly journals Expression and Characterization of a Mycoplasma genitalium Glycosyltransferase in Membrane Glycolipid Biosynthesis

2011 ◽  
Vol 286 (41) ◽  
pp. 35367-35379 ◽  
Author(s):  
Eduardo Andrés ◽  
Núria Martínez ◽  
Antoni Planas
Keyword(s):  
Plasma Membrane ◽  
Essential Gene ◽  
Expression System ◽  
Mycoplasma Genitalium ◽  
Cell Growth Inhibition ◽  
Anionic Phospholipid ◽  
Order Of Magnitude ◽  
Galactosyl Residues

Mycoplasmas contain glycoglycerolipids in their plasma membrane as key structural components involved in bilayer properties and stability. A membrane-associated glycosyltransferase (GT), GT MG517, has been identified in Mycoplasma genitalium, which sequentially produces monoglycosyl- and diglycosyldiacylglycerols. When recombinantly expressed in Escherichia coli, the enzyme was functional in vivo and yielded membrane glycolipids from which Glcβ1,6GlcβDAG was identified as the main product. A chaperone co-expression system and extraction with CHAPS detergent afforded soluble protein that was purified by affinity chromatography. GT MG517 transfers glucosyl and galactosyl residues from UDP-Glc and UDP-Gal to dioleoylglycerol (DOG) acceptor to form the corresponding β-glycosyl-DOG, which then acts as acceptor to give β-diglycosyl-DOG products. The enzyme (GT2 family) follows Michaelis-Menten kinetics. kcat is about 5-fold higher for UDP-Gal with either DOG or monoglucosyldioleoylglycerol acceptors, but it shows better binding for UDP-Glc than UDP-Gal, as reflected by the lower Km, which results in similar kcat/Km values for both donors. Although sequentially adding glycosyl residues with β-1,6 connectivity, the first glycosyltransferase activity (to DOG) is about 1 order of magnitude higher than the second (to monoglucosyldioleoylglycerol). Because the ratio between the non-bilayer-forming monoglycosyldiacylglycerols and the bilayer-prone diglycosyldiacylglycerols contributes to regulate the properties of the plasma membrane, both synthase activities are probably regulated. Dioleoylphosphatidylglycerol (anionic phospholipid) activates the enzyme, kcat linearly increasing with dioleoylphosphatidylglycerol concentration. GT MG517 is shown to be encoded by an essential gene, and the addition of GT inhibitors results in cell growth inhibition. It is proposed that glycolipid synthases are potential targets for drug discovery against infections by mycoplasmas.

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