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

Forty-nine regions in 21 proteins were identified as potential heparin-binding sites based on the sequence organizations of their basic and nonbasic residues. Twelve known heparin-binding sequences in vitronectin, apolipoproteins E and B-100, and platelet factor 4 were used to formulate two search strings for identifying potential heparin-binding regions in other proteins. Consensus sequences for glycosaminoglycan recognition were determined as [-X-B-B-X-B-X-] and [-X-B-B-B-X-X-B-X-] where B is the probability of a basic residue and X is a hydropathic residue. Predictions were then made as to the heparin-binding domains in endothelial cell growth factor, purpurin, and antithrombin-III. Many of the natural sequences conforming to these consensus motifs show prominent amphipathic periodicities having both alpha-helical and beta-strand conformations as determined by predictive algorithms and circular dichroism studies. The heparin-binding domain of vitronectin was modeled and formed a hydrophilic pocket that wrapped around and folded over a heparin octasaccharide, yielding a complementary structure. We suggest that these consensus sequence elements form potential nucleation sites for the recognition of polyanions in proteins and may provide a useful guide in identifying heparin-binding regions in other proteins. The possible relevance of protein-glycosaminoglycans interactions in atherosclerosis is discussed.

https://www.ahajournals.org/doi/pdf/10.1161/01.ATV.9.1.21

Molecular modeling of protein-glycosaminoglycan interactions.

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.

Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix Nancy Boudreau,* Carolyn J. Sympson, Zena Werb, Mina J. Bissell N. Boudreau and M. J. Bissell Life Sciences Division, Lawrence Berkeley Laboratory 1 Cyclotron Road, Building 83, Berkeley, CA 94720, USA. C. J. Sympson Life Sciences Division, Lawrence Berkeley Laboratory 1 Cyclotron Road, Building 83, Berkeley, CA 94720, USA Laboratory of Radiobiology and Environmental Health University of California, San Francisco, CA 94143, USA. Z. Werb Laboratory of Radiobiology and Environmental Health University of California, San Francisco, CA 94143, USA. *To whom correspondence should be addressed. LBNL/DOE funding & contract number: DE-AC02-05CH11231 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California.

Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

Complications of atherosclerosis are the most common cause of death in Western societies. Among the many risk factors identified by epidemiological studies, only elevated levels of lipoproteins containing apolipoprotein (apo) B can drive the development of atherosclerosis in humans and experimental animals even in the absence of other risk factors. However, the mechanisms that lead to atherosclerosis are still poorly understood. We tested the hypothesis that the subendothelial retention of atherogenic apoB-containing lipoproteins is the initiating event in atherogenesis. The extracellular matrix of the subendothelium, particularly proteoglycans, is thought to play a major role in the retention of atherogenic lipoproteins. The interaction between atherogenic lipoproteins and proteoglycans involves an ionic interaction between basic amino acids in apoB100 and negatively charged sulphate groups on the proteoglycans. Here we present direct experimental evidence that the atherogenicity of apoB-containing low-density lipoproteins (LDL) is linked to their affinity for artery wall proteoglycans. Mice expressing proteoglycan-binding-defective LDL developed significantly less atherosclerosis than mice expressing wild-type control LDL. We conclude that subendothelial retention of apoB100-containing lipoprotein is an early step in atherogenesis.

Subendothelial retention of atherogenic lipoproteins in early atherosclerosis.

Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris

Apolipoprotein B-100 (apoB-100) is the major protein constituent of human plasma low-density lipoproteins (LDL). On the basis of its amino acid sequence [Chen, S.-H., Yang, C.-Y., Chen, P.-F., Setzer, D., Tanimura, M., Li, W.-H., Gotto, A. M., Jr., & Chan, L. (1986) J. Biol. Chem. 261, 12918-12921], apo B-100 is one of the largest monomeric proteins known with a calculated molecular weight of 512937. Heparin binds to the LDL surface by interacting with positively charged amino acid residues of apoB-100, forming soluble complexes in the absence of divalent metals and insoluble complexes in their presence. The purpose of this study was to isolate and characterize the heparin-binding domain(s) of apoB-100. Human plasma LDL were fragmented with cyanogen bromide (CNBr). After delipidation and reduction-carboxymethylation, the CNBr peptides were fractionated by sequential chromatography on DEAE-Sephacel, Mono S, and high reactive heparin (HRH) AffiGel-10; HRH was purified by chromatography of crude bovine lung heparin on LDL AffiGel-10. Heparin-binding peptides were further purified by reverse-phase high-performance liquid chromatography. Heparin-binding activity was monitored by a dot-blot assay with 125I-HRH. The amino-terminal sequences of four CNBr heparin-binding peptides (CNBr-I-IV) were determined. CNBr-I-IV correspond to residues 2016-2151, 3109-3240, 3308-3394, and 3570-3719, respectively, of the amino acid sequence of apoB-100. Each CNBr peptide contains a domain(s) of basic amino acid residues which we suggest accounts for their heparin-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B.

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

The complete covalent structure of dihydrofolate reductase from chicken liver is described. The S-carboxymethylated protein was subjected to cleavage by cyanogen bromide which produced five fragments. Fragment CB2 contained an internal homoserine residue which was not cleaved by cyanogen bromide. Sequences and ordering of the cyanogen bromide fragments were established by means of automated sequencer analyses of the fragments and from smaller peptides generated by proteolysis with trypsin and staphylococcal protease. The covalent structure of the single polypeptide chain comprises 189 residues of molecular weight 21,651. The chicken liver enzyme is homologous to that from L1210 cells and shows regions of homology to dihydrofolate reductases from Streptococcus faecium, Escherichia coli, and Lactobacillus casei. These homologous regions in the chicken liver enzyme are primarily related to conserved amino acid residues implicated in the binding of NADPH and methotrexate by bacterial dihydrofolate reductases.

Primary structure of chicken liver dihydrofolate reductase.

Dihydrofolate reductase has been isolated from methotrexate-resistant WIL2 human lymphoblastoid cells. This subline produces ca. 150 times more enzyme than the parental drug-sensitive line. The reductase has been purified to homogeneity by methotrexate affinity chromatography, gel filtration, and preparative isoelectric focusing in a yield of 65%. The enzyme has a pI = 7.7 and a molecular weight of ca. 22000. The amino-terminal 27 amino acid residues have been determined, revealing the location of the single cysteine residue at position 6. Reaction of this cysteine with p-(hydroxy-mercuri)benzoate in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) results in a 5-fold increase in enzyme activity. Other agents including KCl, urea, and thiourea also cause enzyme activation. The Km values for NADPH and dihydrofolate are 0.25 and 0.036 microM, respectively. Mercurial activation of the enzyme results in a 27-fold increase in the Km for NADPH and a 35-fold increase in the Km for dihydrofolate.

Dale T. Blankenship

Papers

Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris

Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B.

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

Primary structure of chicken liver dihydrofolate reductase.

Purification and characterization of dihydrofolate reductase from methotrexate-resistant human lymphoblastoid cells.