Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.     

A New Role for Escherichia coli DsbC Protein in Protection against Oxidative Stress

We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the l-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing l-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress.     

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.     

Disulfide-dependent folding and export of Escherichia coli DsbC

DsbC, a member of the Dsb family in the periplasm of Gram-negative bacteria, is not only a disulfide isomerase but also a chaperone. Five DsbC mutants with Cys in the active site sequence of Cys(98)-Gly-Tyr-Cys(101) and the nonactive site disulfide Cys(141)-Cys(163) replaced by Ser have been studied. The results show that the active site Cys residues are necessary for enzyme activities but not required for chaperone activity, while the lack of the nonactive site disulfide results in a decreased chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase but has no effect on enzyme activities. Wild-type DsbC was overexpressed and correctly processed as a soluble periplasmic protein. Mutation in one of these Cys residues results in aggregation or extracellular/membrane locations, but does not affect the proper processing. DsbC mutated in either Cys residue of nonactive site disulfide shows higher sensitivity to unfolding by guanidine hydrochloride and slower refolding compared with wild-type DsbC and the active site Cys mutants. The above results provide experimental evidence for structural role of the nonactive site disulfide in folding and biological activities of DsbC.     

用质粒pUC18在大肠杆菌中表达人蛋白质二硫键异构酶

 用质粒ｐＵＣ１８在大肠杆菌中表达人蛋白质二硫键异构酶高音,王志珍（中国科学院生物物理研究所,生物大分子国家重点实验室,北京１００１０１）蛋白质二硫键异构酶（ｐｒｏｔｅｉｎｄｉｓｕｌｆｉｄｅｉｓｏｍｅｒａｓｅ,ＰＤＩ）催化蛋白质分子内天然二硫键的形成,...     

重组二硫键异构酶在大肠杆菌中表达条件的统计优化

二硫键形成蛋白A (disulfidebondformationproteinA ,DsbA)是大肠杆菌周质胞腔中辅助多种含有二硫键的蛋白质正确折叠并具有生物学活性的一种二硫键异构酶.通过统计实验设计的方法将生产重组DsbA的发酵过程进行了优化.首先通过Plackett Burman设计挑选出了对DsbA表达量影响较大的四个因素,然后再利用杂合设计进行实验,并通过拟合得到了响应曲面函数,但该函数的驻点是鞍点,因此不具有全局的极值.最后通过约束优化得到了较佳的实验点,在该实验点下DsbA的表达量比基本培养条件下提高了5 0 .14 % .     

Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b''a'' domain of protein disulfide isomerase (PDIb''a'') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb''a'' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.     

Autodegradation of Protein Disulfide Isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.     

Dimerization of the RelA protein of Escherichia coli.

The RelA protein of Escherichia coli is a ribosome-associated (p)ppGpp synthetase that is activated by amino acid deprivation. It was recently reported that the activity of RelA is regulated by oligomerization mediated by the C-terminal domain of RelA. The oligomerization of RelA is further characterized in this study. The C-terminal domain consisting of amino acids 455-744, designated 'RelA, formed homooligomers as well as heterooligomers with RelA as demonstrated by copurification of RelA and 'RelA and by an affinity blotting assay. Glutaraldehyde-induced cross-linking indicated that the oligomer was a dimer. The functional analysis of 'RelA was based on a combination of yeast two-hybrid analysis, the determination of the effects of overexpression of 'RelA derivatives on the stringent response, and the cellular localization of the overexpressed 'RelA derivatives. These studies indicated that two regions, designated 'RelA-1 (amino acids 455-538) and 'RelA-2 (amino acids 550-682), were involved in dimerization. The involvement of one of these two regions, RelA-2, is consistent with a previous site-directed mutagenesis study. In addition to dimerization, 'RelA-2 apparently contained the main ribosome-binding domain of RelA. The third region, 'RelA-3 (amino acids 682-744), was not involved in either dimerization or ribosome binding. The overexpression of 'RelA-1 and 'RelA-2, but not 'RelA-3, inhibited the stringent response. These results support the previously proposed model which suggests a role for oligomerization in the regulation of (p)ppGpp synthetase.     

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.     

Overexpression of Protein Disulfide Isomerase in Aspergillus

One of the major problems with the production of biotechnologically valuable proteins has been the purification of the product. For Escherichia coli and Saccharomyces cerevisiae, there are several techniques for the purification of intracellular proteins, but these are time consuming and often result in poor yields. Purification can be considerably facilitated, if the product is secreted from the host cell. In the work presented, we have constructed an expression vector (pSGNH2) for the secretion of protein disulfide isomerase (PDI; EC 5.3.4.1) from Aspergillus niger, in which the retention signal His-Asp-Glu-Leu (H-D-E-L) was modified to Ala-Leu-Glu-Gln (A-L-E-Q) via the polymerase chain reaction (PCR) method. The PDI gene was placed under the control of the A. oryzaeα-amylase promoter. This expression vector was transformed into A. niger NRRL3, resulting in PDI secretion into the medium. The catalytic activity of overexpressed PDI from A. niger was indistinguishable from that of PDI isolated from bovine liver. With further strain improvement and optimization of culture conditions, it could be possible to raise the PDI production to the bioprocessing scale. Received: 26 April 2000 / Accepted: 30 May 2000     

Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 x 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 A resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.     

Folding of Escherichia coli DsbC: characterization of a monomeric folding intermediate

The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC was thermodynamically reversible. The equilibrium folding profile measured by fluorescence excited at 280 nm exhibited a three-state transition profile with a stable folding intermediate formed at 0-2.0 M GdnHCl followed by a second transition at higher GdnHCl concentrations. Sedimentation velocity data revealed dissociation of the dimer to the monomer over the concentration range of the first transition (0-2.0 M). In contrast, fluorescence emission data for DsbC excited at 295 nm showed a single two-state transition. Fluorescence emission data for the equilibrium unfolding of the monomeric G49R mutant, excited at either 295 or 280 nm, indicated a single two-state transition. Data obtained for the dimeric Y52W mutant indicated a strong protein concentration dependence of the first transition but no dependence of the second transition in equilibrium unfolding. This suggests that the fluorescence of Y52W sensitively reports conformational changes caused by dissociation of the dimer. Thus, the folding of DsbC follows a three-state transition model with a monomeric folding intermediate formed in 0-2.0 M GdnHCl. The folding of DsbC in the presence of DTT indicates an important role for the non-active site disulfide bond in stabilizing the conformation of the molecule. Dimerization ensures the performance of chaperone and isomerase functions of DsbC.     

Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC

In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC- strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC- strain, even though the dsbA- single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.     

Compact Conformations of Human Protein Disulfide Isomerase

Protein disulfide isomerase (PDI) composed of four thioredoxin-like domains a, b, b'', and a'', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI) in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b'' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a'' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.     

人蛋白质二硫键异构酶Cdna基因的克隆及在大肠杆菌中的表达

从人胚胎肝组织中分离纯化出总RNA,在总RNA中提取mRNA,经逆转录得到cDNA第一条链,并以之为模板和相应寡聚脱氧核苷酸为引物．进行PCR合成人蛋白质二硫键异构酶(Proteim disulfide isomerase,PDI)cDNA基因,将所得cDNA克隆到质粒pUCl8上,转化大肠杆菌DH5a细胞,进行诱导表达筛选和活性筛选。将筛选的基因克隆到载体pBV220上,在大肠杆菌细胞中表达,产物以溶解形式在胞浆中表达。表达产物经硫酸铵分级沉淀和DEAE Sepharose Fast Flow柱层析分离,产物的纯度与活性分别为90％、1100u／g。     

大肠杆菌甘露糖异构酶(MI)基因克隆及功能研究

甘露糖异构酶(EC 5.3.1.7,Mannose isomerase,简称MI)能够催化果糖和甘露糖之间的异构转化.甘露糖加氢催化形成甘露醇,因此甘露糖异构酶的研究为工业生产甘露糖和甘露醇提供了新的可能途径.本研究以Escherichia coli JM109基因组DNA为模板,PCR扩增mi基因.结果表明,所克隆的mi基因与E.coli str.K-12 substr.MG1655的MI基因序列一致性为99.9%,氨基酸序列一致性为99%.该基因能够在E.coli BL21(DE3)中进行高效诱导表达,诱导出51.4 kD的特异性融合蛋白.酶学研究表明,该酶最适反应温度为37℃,最适pH为7.5,甘露糖为最佳反应底物,反应平衡时果糖与甘露糖的比值约为2.7.     

Expression and Localization of Plant Protein Disulfide Isomerase

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.