Encoding Protein Disulfide Isomerase Research Articles

A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material

BackgroundKomagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers.ResultsHere, we describe a linear “nano”chromosome (of 15–25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of PAOX1 and demonstrated that the secreted protein was active as a regulator of the complement system.ConclusionsWe have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.

Essential cell-extrinsic requirement for PDIA6 in lymphoid and myeloid development.

In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell-independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.

High-level expression of Thermomyces dupontii thermophilic lipase in Pichia pastoris via combined strategies.

In this study, a combined strategy was used to improve the production of Thermomyces dupontii lipase (TDL) in Pichia pastoris. First, the native gene of TDL was optimized based on the codon usage of P. pastoris, ligated to pPICZαA and transformed in P. pastoris X33. A recombinant strain designated X33-T23 with the highest activity (1020 U/mL in shake flasks) amongst 216 recombinant colonies was selected for further investigations. To further increase the production of TDL, nine different secretion helper factor genes were transformed in the recombinant strain, X33-T23. The recombinant strain co-expression with the gene encoding protein disulfide isomerase, designated X33-T23-PDI, exhibited the highest activity in shake flasks (1760 U/mL) and in 5L bioreactor (57521 U/mL) which were 1.67- and 1.46-fold higher, respectively, than for strain X33-T23. Additionally, the optimization of the inducers (temperature and pH) for the recombinant strain X33-T23-PDI in 5L bioreactor produced, as expected, much higher lipase activity (81203 U/mL). The results of this study will provide an effective method to produce TDL and give some clues on how to improve production of heterologous proteins in P. pastoris.

\u201cExpression of genes encoding protein disulfide isomerase (PDI) in cultivars and lines of common wheat with different baking quality of flour\u201d

BackgroundThe subject of this research was to investigate the level of expression of genes encoding protein disulfide isomerase (PDI) in cultivars and lines of wheat with different baking value of flour. PDI plays a key role in the formation of disulfide bonds in newly formed proteins. Each of cultivars and lines had a specific set of high molecular weight glutenin subunits (HMW-GS). Based on the presence of individual subunits, the potential baking value is predicted. Sometimes this value is not confirmed during technological analysis. Since there are cases where flour has a better or worse value than expected on the basis of the genotype, the expression of PDI genes was considered as a potential cause for discrepancies mentioned.ResultsAnalysis focused on three stages of grain development. The expression level of PDI genes was compared between wheat cultivars and lines with different genotype-phenotype combinations, which means diversified sets of HMW-GS combined with diversified qualitative classification. The highest expression level of PDI was noticed at early stage of grain development, which is consistent with the function of PDI. The expression level was evaluated by the real-time PCR technique.ConclusionResults obtained in this work did not allow for a clear statement of decisive significance of PDI in the context of shaping the final baking value. The results of this work contribute to an ever more in-depth understanding of the mechanisms governing baking value, and thus to the progress of the selection of new varieties with more beneficial properties.

Characterization of Genes Encoding Protein Disulfide Isomerase in Wheat Including the Specific Role of PDI in the Formation of Gluten.

The results of phylogenetic analyses revealed that the family of plant PDI may comprise of at least eight different subfamilies with varying numbers and positions of active centers while retention signals in the endoplasmic reticulum may be present or absent. At least one gene has been cloned for each phylogenetic group. Other phylogenetic analyses have indicated that the family of PDIlike proteins consists of ten classes, the first five of which include proteins equipped with two thioredoxin domains. These results indicate complexity and diversity of the family of protein disulfide isomerase in plants. The study of molecular characteristics of PDI in some cereal species have shown that this enzyme participates in the maturation of secretory proteins and also in the formation of albuminous substances in endosperm, in the mechanism of formation of disulfide bonds and polymerization of gluten polypeptides in wheat. The mechanism of formation of disulfide linkage was tested through in vitro experiments. However, it is not entirely certain whether it reflects their formation in vivo. The results of researches suggest that protein folding and disulfide bond formation occurs in the endoplasmic reticulum. PDI plays a prominent role among enzymes involved in posttranslational modification of proteins. The main goal of this work is to present research data on protein disulfide isomerase, which may be a leading research objective in the area of wheat gluten and the impact of PDI on the baking quality of wheat flour.

Molecular Cloning, Expression Analysis, and Preliminarily Functional Characterization of the Gene Encoding Protein Disulfide Isomerase from Jatropha curcas.

Reactive oxygen species (ROS) in plants, arising from various environmental stresses, impair the thiol-contained proteins that are susceptible to irregular oxidative formation of disulfide bonds, which might be alleviated by a relatively specific modifier called protein disulfide isomerase (PDI). From our previous data of the transcriptome and digital gene expression of cold-hardened Jatropha curcas, a PDI gene was proposed to be cold-relevant. In this study, its full-length cDNA (JcPDI) was cloned, with the size of 1649 bp containing the entire open reading frame (ORF) of 1515 bp. This ORF encodes a polypeptide of 504 amino acids with theoretical molecular weight of 56.6 kDa and pI value of 4.85. One N-terminal signal peptide (-MASKGSIWSCMFLFSLI VAISAGEG-) and the C-terminal anchoring sequence motif (-KDEL-) specific to the endoplasmic reticulum, as well as two thioredoxin domains (-CGHC-), are also found by predictions. Through semi-quantitative RT-PCR, the expression of JcPDI was characterized to be tissue-differential strongly in leaves and roots, but weakly in stems, and of cold-induced alternations. Furthermore, JcPDI overexpression in yeast could notably enhance the cold resistance of host cells. Conclusively, these results explicitly suggested a considerable association of JcPDI to cold response and a putative application potential for its correlated genetic engineering.

A second protein disulfide isomerase plays a protective role against nitrosative and nutritional stresses in Schizosaccharomyces pombe

In the present work, a second gene encoding protein disulfide isomerase (PDI2) was cloned and characterized from Schizosaccharomyces pombe, and its regulation was studied. The structural gene encoding PDI2 was amplified from the genomic DNA using PCR, and ligated into the E. coli-yeast shuttle vector pRS316 to generate the recombinant plasmid pYPDI2. The determined DNA sequence carries 2,578 bp and is able to encode a protein of 726 amino acid sequence with CGAC at the putative active site. The fission yeast cells harboring pYPDI2 contained 1.62- and 2.73-fold higher PDI activity than the control yeast cells in exponential and stationary phases, respectively, indicating that the cloned gene is in vivo functioning. The PDI2 mRNA levels in both vector control and pYPDI2-containing yeast cells were found to be significantly higher in the stationary phase than in the exponential phase, suggesting that expression of the PDI2 gene is under stationary control. The yeast cells harboring pYPDI2 showed enhanced survival on minimal media plates containing nitric oxide (NO)-generating sodium nitroprusside (SNP) and no nitrogen. The synthesis of β-galactosidase from the PDI2-lacZ fusion gene was markedly enhanced in the Pap1-positive KP1 cells by SNP and nitrogen starvation. However, the enhancement in the synthesis of β-galactosidase from the PDI2-lacZ fusion gene by SNP and nitrogen starvation appeared to be relatively reduced in the Pap1-negative TP108-3C cells than in the Pap1-positive KP1 cells. The PDI2 mRNA level was elevated by SNP and nitrogen starvation in the Pap1-positive cells but not in the Pap1-negative cells. In brief, the S. pombe PDI2 plays a protective role against nitrosative and nutritional stresses, and is positively regulated by NO and nitrogen starvation in a Pap1-dependent manner.

Expression of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae using GAL1 promoter

Hepatitis B virus, a member of the hepadnavirus family, causes the acute and chronic diseases of the human liver. The S domain of hepatitis B virus surface antigen (sHBsAg) was expressed under the control of the galactose-inducible GAL1 promoter in recombinant Saccharomyces cerevisiae. Batch fermentation of S. cerevisiae 2805/pδMFα-sHBsAg resulted in 4.92 g l −1 dry cell mass and 2.21 mg l −1 sHBsAg concentration. To improve the expression level of sHBsAg, the pdi1 gene encoding protein disulfide isomerase was coexpressed in recombinant S. cerevisiae. Batch fermentation of S. cerevisiae 2805/pδMFα-sHBsAg + pPDI using 21 g l −1 glucose and 33 g l −1 galactose resulted in 9.75 g l −1 dry cell mass and 13.3 mg l −1 sHBsAg concentration, which were 2 and 6 times higher than those for S. cerevisiae 2805/pδMFα-sHBsAg, respectively. Appearance of three sHBsAgs with different molecular sizes of 24 kDa, 34 kDa and 40 kDa in immunoblot assay and their endoglycosidase treatment indicated that sHBsAg might be expressed in three types of the authentic, MFα signal sequence-containing and N-glycosylated MFα signal sequence-containing forms. Fed-batch fermentation of recombinant S. cerevisiae 2805 coexpressing the sHBsAg and pdi1 genes was carried out by feeding 600 g l −1 glucose continuously and controlling galactose concentration at around 20 g l −1. As a result, 20.2 g l −1 dry cell mass and 74.4 mg l −1 maximum sHBsAg concentration were obtained, which were 4.1 and 33.7 times higher than those for the batch fermentation of S. cerevisiae 2805/pδMFα-sHBsAg, respectively.

A protein disulfide isomerase expressed in the embryonic midline is required for left/right asymmetries.

Although the vertebrate embryonic midline plays a critical role in determining the left/right asymmetric development of multiple organs, few genes expressed in the midline are known to function specifically in establishing laterality patterning. Here we show that a gene encoding protein disulfide isomerase P5 (PDI-P5) is expressed at high levels in the organizer and axial mesoderm and is required for establishing left/right asymmetries in the zebrafish embryo. pdi-p5 was discovered in a screen to detect genes down-regulated in the zebrafish midline mutant one-eyed pinhead and expressed predominantly in midline tissues of wild-type embryos. Depletion of the pdi-p5 product with morpholino antisense oligonucleotides results in loss of the asymmetric development of the heart, liver, pancreas, and gut. In addition, PDI-P5 depletion results in bilateral expression of all genes known to be expressed asymmetrically in the lateral plate mesoderm and the brain during embryogenesis. The laterality defects caused by pdi-p5 antisense treatment arise solely due to loss of the PDI-P5 protein, as they are reversed when treated embryos are supplied with an exogenous source of the PDI-P5 protein. Thus the spectrum of laterality defects resulting from depletion of the PDI-P5 protein fully recapitulates that resulting from loss of the midline. As loss of PDI-P5 does not appear to interfere with other aspects of midline development or function, we propose that PDI-P5 is specifically involved in the production of midline-derived signals required to establish left/right asymmetry.

Functional differences in yeast protein disulfide isomerases.

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

Developmental localization of retina cognin synthesis by in situ hybridization

Retina cognin (R-cognin) is a 50 kDa protein involved in cell recognition and neuronal differentiation during development of the embryonic chick retina. Initial characterization of a partial cDNA encoding R-cognin revealed a striking similarity to the cDNA encoding protein disulfide isomerase (PDI), a 57 kDa multifunctional protein. The exact nature of the relationship between R-cognin and PDI is not known; however, both proteins appear to be encoded by the same gene. In the present study, we developed cRNA probes to examine the expression of R-cognin and PDI transcripts in embryonic chick retina and liver. In the retina, the amount of transcript decreased with embryonic age, in parallel to a similar decrease in R-cognin protein. In the liver, where PDI is prominently expressed, the amount of transcript was not developmentally regulated. The spatial and temporal pattern of expression of the R-cognin-encoding retinal transcript was examined by in situ hybridization. R-cognin mRNA was expressed in cells across the retina early in retinogenesis, but became restricted to the cells of the inner retina later in development. This pattern of expression was the same as the developmental pattern of R-cognin protein [Dobi et al., Invest. Ophthalmol. Vis. Sci. 27, (1986) p. 323–329], thus, demonstrating that this secreted protein functions at the surface of the cells where it is transcribed.

Cloning, characterization and overexpression of a gene ( pdiA) encoding protein disulfide isomerase of Aspergillus oryzae

We amplified a 1.1-kb fragment of DNA from genomic DNA of Aspergillus oryzae using the polymerasechain reaction (PCR) and primers, the sequences of which correspond to the consensus sequences coding for two thioredoxin domains of protein disulfide isomerase (PDI). A genomic DNA clone of A. oryzae was isolated by hybridization with the amplified DNA fragment. Southern hybridization analysis revealed that the gene encoding PDI ( pdiA) was present in a 5.0-kb XhoI region of the A. oryzae chromosome as a single copy. pdiA contains three putative introns and encodes 515 amino acid residues. The deduced amino acid sequence of A. oryzae PDI exhibits a high degree of homology (51%) with Humicola insolens PDI, while it exhibits less than 20% homology with those of yeast, plants, and mammals. The amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum (ER) retention signal, HDEL, and two putative active site sequences (WCGHCK) of PDI. When the cloned pdiA was expressed under the control of the amyB promoter in an A. oryzae transformant, the PDI activity in the microsomal fraction was about 10-fold higher than that in a microsomal fraction of the recipient strain, indicating that the cloned gene encodes functional PDI.

Nucleotide sequence of a wheat cDNA encoding protein disulfide isomerase.

PDI (EC 5.3.4.1) is an ER-resident protein involved in the rearrangement of disulfide bonds of secretory proteins. This protein also appears as one of the two subunits of another ER resident protein, prolyl 4-hydroxylase (EC 1.14.11.2), and may be involved in various cellular processes (Noiva and Lennarz, 1992). PDI appears to be very abundant in developing wheat grains (Roden et al., 1982) and was also demonstrated to catalyze intrachain disulfide bond formation in a wheat y-gliadin storage protein (Bulleid and Freedman, 1988). To analyze the expression of PDI in developing grains and to study further its role in the folding and assembly of wheat storage proteins, we have cloned a full-length wheat PDI cDNA by screening a root-tip cDNA library using maize PDI as a DNA probe (Table I). Sequence analysis of the wheat cDNA revealed an open reading frame encoding a protein of 515 amino acids, which also exhibited 96 and 63% amino acid sequence identity with full-length PDI cDNAs from barley (accession No. L33250) and alfalfa (Shorrosh and Dixon, 1991), respectively. The deduced amino acid sequence of the wheat PDI also contained an N-terminal signal peptide of 25 amino acids, two conserved thioredoxin-like active sites, a potential N-linked glycosylation site, as well as a C-terminal KDEL sequence generally present in ER-resident proteins.

Nucleotide sequence and developmental expression of duplicated genes encoding protein disulfide isomerase in barley (Hordeum vulgare L.).

PDI (EC 5.3.4.1), an ER luminal protein that catalyzes the formation of disulfide bonds of nascent polypeptide chains in the lumen of ER (Freedman, 1989), is a multifunctional protein that also functions as the p subunit of prolyl 4hydroxylase (Pihlajaniemi et al., 1987), a thyroid hormonebinding protein (Yamauchi et al., 1987), and a component of the triglyceride transfer protein complex (Wetterau et al., 1990). In addition, the amino acid sequence of PDI is similar to a number of enzymes (Bennett et al., 1988; Geetha-Habib et al., 1988; Tsibris et al., 1989). PDI contains two copies of an active site closely related to thioredoxin. A cDNA sequence for plant PDI has been reported in alfalfa (Medicago sativa) (Shorrosh and Dixon, 1991, 1992); however, here we report three different cDNA clones encoding PDI in barley (Hordeum vulgare). A cDNA library in Uni-ZAP XR was constructed from poly(A)+ RNA of barley ovaries 3 d after pollination. Three different cDNA clones (PDIl, PDI2, and PDI3) were identified in the course of differential screening to characterize fertilization-specific gene expression. The DNA sequence of each clone was determined by the dideoxy method of Sanger with an autosequencer (Applied Biosystems, Inc. [Foster City, CAI model 373A, version 1.2.0). PDI2 was used as a probe for restriction fragment-length polymorphism mapping in two barley doubled-haploid populations. PDIl (1816 bp) and PDI2 (1772 bp) contained a complete coding sequence, but PD13 (1191 bp) was truncated at the 5' region. The three cDNA clones differed at the 3' noncoding region before the poly(A) tail and were likely transcribed from different PDI loci. Two independent PDI loci were detected in 150 doubled-haploid progeny of Harrington X Morex. One of these loci was mapped to the plus arm of barley chromosome 1, which is 2.7 centimorgans from the restriction fragment-length polymorphism marker ABC154a in the barley genome mapping population of Steptoe X Morex (Kleinhofs et al., 1993). Additional PDI loci are likely since multiple bands were detected in DNA genomic blots. Multiple cDNA sequences and PDI loci indicate the presence of a small PDI gene family in barley.

Molecular Structure of a Yeast Gene, PDI1, Encoding Protein Disulfide Isomerase That Is Essential for Cell Growth

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.