During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

http://www.biotechlab.ch/UserFiles/File/787339_Ref_CererghinoCregg2000.pdf

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

/pdf/heterologous-protein-production-using-the-pichia-pastoris-22lqcx2i7v.pdf

Heterologous protein production using the Pichia pastoris expression system.

The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.

Recent advances in the expression of foreign genes in Pichia pastoris

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and can be divided into two groups: muscle receptors, which are found at the skeletal neuromuscular junction where they mediate neuromuscular transmission, and neuronal receptors, which are found throughout the peripheral and central nervous system where they are involved in fast synaptic transmission. nAChRs are pentameric structures that are made up of combinations of individual subunits. Twelve neuronal nAChR subunits have been described, alpha2-alpha10 and beta2-beta4; these are differentially expressed throughout the nervous system and combine to form nAChRs with a wide range of physiological and pharmacological profiles. The nAChR has been proposed as a model of an allosteric protein in which effects arising from the binding of a ligand to a site on the protein can lead to changes in another part of the molecule. A great deal is known about the structure of the pentameric receptor. The extracellular domain contains binding sites for numerous ligands, which alter receptor behavior through allosteric mechanisms. Functional studies have revealed that nAChRs contribute to the control of resting membrane potential, modulation of synaptic transmission and mediation of fast excitatory transmission. To date, ten genes have been identified in the human genome coding for the nAChRs. nAChRs have been demonstrated to be involved in cognitive processes such as learning and memory and control of movement in normal subjects. Recent data from knockout animals has extended the understanding of nAChR function. Dysfunction of nAChR has been linked to a number of human diseases such as schizophrenia, Alzheimer's and Parkinson's diseases. nAChRs also play a significant role in nicotine addiction, which is a major public health concern. A genetically transmissible epilepsy, ADNFLE, has been associated with specific mutations in the gene coding for the alpha4 or beta2 subunits, which leads to altered receptor properties.

Nicotinic acetylcholine receptors: from structure to brain function.

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 FUNCTIONS OF PEROXISOMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' 447 HUMAN PEROXISOMAL DISORDERS . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 PEROXISOMES AS A MODEL FOR ORGANELLE ASSEMBLY AND DISASSEMBLY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 1 Peroxisome Import and Assembly Mutants . . . . . . . . . . . . . . . . . . . . . . . . 451 PROTEIN IMPORT INTO PEROXISOMES . . . . . . . . . . . . . . . . . . . . . . . . . 454 Targeting of Matrix Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455 Conservation of the PTSI Tripeptide Variants . . . . . . . . . . . . . . . . . . . . . . 457 An Amino-Terminal PTS . . . . .. . . . . . . . . . . . .. . . .. . ... . . . . . . . . 458 Other Peroxisomal Matrix Targeting Signals . . . . . . . . . . . . . . . . . . . . . . . 459 Evolutionary Use of Diff erent PTSs by the Same Protein . . . . . . . . . . . . . . . 459 Targeting Signals for Peroxisomal Membrane Proteins . . . . . . . . . .. . . . . . 460 Receptors for PTSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460 Peroxisomal Protein Import Deficiencies in Human Disease . . . . . . . . . . . . . 461 Mutations Responsible for Some Human Peroxisomal Disorders . . . . . . . . . . . 462 APPROACHES USED TO ELUCIDATE THE MECHANISM OF PROTEIN IMPORT INTO PEROXISOMES . . . . . . . . . . . . . . . . . . . . . . . . . 463 In Vitro Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 Microinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 Semi-intact Cells . ... . . . . . . . . . . . . . . . . . .... . . . .. . . . . . . . . . . 464 Mechanistic Aspects of Protein Import Into the Peroxisomal Matrix . . . . . . .. 464 DISTINCT STEPS IN PEROXISOME BIOGENESIS . . . . . . . . . . . . . . . .. . . 465 GENERAL PROPERTIES OF PEROXISOME ASSEMBLY/IMPORT MUTANTS . 468 Mutants Affected in Peroxisome Proliferation . . . . . . . . . . . . . . . . . , 468 Mutants Affected in Peroxisome Growth . . . . .... . . . ...... . . . . , . . . . 468

Protein import into peroxisomes and biogenesis of the organelle.

We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.

/pdf/the-hansenula-polymorpha-per8-gene-encodes-a-novel-4vrxpmb65v.pdf

The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation.

PER3, a Gene Required for Peroxisome Biogenesis in Pichia pastoris, Encodes a Peroxisomal Membrane Protein Involved in Protein Import

We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis.

/pdf/an-efficient-screen-for-peroxisome-deficient-mutants-of-27c1mfnayv.pdf

An efficient screen for peroxisome-deficient mutants of Pichia pastoris.

In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut-) mutants are affected in genes required for peroxisome biogenesis (PER genes). Previously, we reported that one group of per mutants, termed Pim-, are characterized by the presence of a few small peroxisomes with the bulk of peroxisomal enzymes located in the cytosol. Here, we describe a second major group of per mutants that were observed to be devoid of any peroxisome-like structure (Per-). In each Per- mutant, the peroxisomal methanol-pathway enzymes alcohol oxidase, catalase and dihydroxyacetone synthase were present and active but located in the cytosol. Together, the Pim- and Per- mutant collections involved mutations in 14 different PER genes. Two of the genes, PER5 and PER7, were represented by both dominant-negative and recessive alleles. Diploids resulting from crosses of dominant per strains and wild-type H. polymorpha were Mut- and harbored peroxisomes with abnormal morphology. This is the first report of dominant-negative mutations affecting peroxisome biogenesis.

/pdf/characterization-of-peroxisome-deficient-mutants-of-sw0v7zel44.pdf

Characterization of peroxisome-deficient mutants of Hansenula polymorpha.

(per) mutantsintheyeast Pichia pastoris. Thescreenrelies on theunusual ability ofP.pastoris togrow on twocarbon sources,methanol andoleic acid, bothofwhichabsolutely require peroxisomes tobemetabolized. A collection of280methanol utilizationdefective (Mut-) P.pastoris mutants was isolated, organized into 46complementation groups,andtested for those that were alsooleate-utilization defective (Out-) butstill capable ofgrowth on ethanol andglucose. Mutants in10groupsmetthis phenotypic description, and8ofthese were observed byelectron microscopyto beperoxisome deficient (Per-). Ineach permutant, Mut-,Out-,andPer-phenotypes weretightly linked and therefore were mostlikely duetoamutation atasingle locus. Subcellular fractionation experiments indicated thattheperoxisomal markerenzyme catalase was mislocalized tothecytosol inbothmethanol- and oleate-induced cultures ofthemutants. Incontrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme,was virtually absent fromper mutantcells. Therelative easeofper mutantisolation inP. pastoris, incolnunction withwell-developed procedures forits molecular andgenetic manipulation, makesthis organism an attractive system forstudies on peroxisome biogenesis. Virtually alleukaryotic cells harborsingle-membraneboundorganelles called peroxisomes. Theyarethesite of hydrogen peroxide-generating oxidative reactions incells and,almost without exception, contain thehemeenzyme catalase tobreakdownthisreactive compound(1,24). Enzymesfoundwithin theperoxisome matrix areinvolved inavariety ofimportant metabolic pathways. However, the specific pathways varysignificantly depending uponthe organism. Inhumans, peroxisomal enzymesareknownto playanessential roleinanumberofanabolic andcatabolic pathways, particularly inlipid metabolism (26). Theimportanceofperoxisomes tohumansisdramatically demonstrated byafamily oflethal genetic disorders called Zellwegersyndrome inwhichperoxisomes appear tobeabsent fromcells (26, 50). Inrecent years, basic features ofperoxisome biogenesis haveemerged. Peroxisomes donotappear tobesynthesized denovoortoarise bybudding fromtheendoplasmic reticulum asdosecretory pathway organelles. Instead, peroxisomes formbybudding frompreexisting peroxisomes (48). Proteins destined forperoxisomal localization aresynthesized oncytosolic orfree ribosomes andareposttranslationally imported (14). Todate, twoperoxisomal targeting signals (PTSs) havebeendefined. A fewperoxisomal enzymes,including rat3-ketoacyl-coenzyme

Xuqiu Tan

Papers

The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation.

PER3, a Gene Required for Peroxisome Biogenesis in Pichia pastoris, Encodes a Peroxisomal Membrane Protein Involved in Protein Import

An efficient screen for peroxisome-deficient mutants of Pichia pastoris.

Characterization of peroxisome-deficient mutants of Hansenula polymorpha.

An Efficient ScreenforPeroxisome-Deficient