Human brain pyridoxal-5'-phosphate phosphatase (PLPP):protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.     

GST-SOD1-R9融合蛋白的表达、纯化、稳定性与跨膜效应

穿膜肽TAT介导的双效抗氧化酶GST(谷胱甘肽巯基转移酶)-TAT-SOD1(Cu,Zn超氧化物歧化酶),可有效清除胞内自由基,其预防氧化损伤的效果强于SOD1-TAT,但前者的跨膜能力不如后者。为增强双效抗氧化酶的跨膜效率,本研究融合了SOD1和穿膜肽R9,合成SOD1-R9全基因序列,并将其插入带有GST的原核表达载体pGEX-4T-1中,成功构建了GST-SOD1-R9融合蛋白表达质粒。然后,将重组质粒pGEX-4T-1-SOD1-R9转化大肠杆菌BL21(DE3),用IPTG诱导表达融合蛋白,通过改变诱导温度和诱导时间,确定了融合蛋白在25℃下表达11 h,可得到高表达量的可溶性GST-SOD1-R9融合蛋白。利用80%硫酸铵沉淀和GST琼脂糖树脂纯化得到纯蛋白,应用SDS-PAGE和酶活性鉴定纯化的蛋白为正确表达的目标蛋白。GST-SOD1-R9融合蛋白的温度和pH稳定性实验结果证实,该蛋白在生理条件下具有良好的SOD和GST活性。细胞跨膜实验结果证明其跨膜能力与GST-TAT-SOD1融合蛋白相比显著增强(P0.05)。这些工作为深入研究GST-SOD1-R9的抗氧化损伤效应建立了基础。     

Expression, purification, and characterization of recombinant NOD1 (NLRC1): A NLR family member

NOD1 (NLRC1) is a member of the NLR family of innate immunity proteins, which are important cellular sensors of various pathogens. Deregulated NOD1 signaling is involved in various autoimmune, inflammatory, and allergic diseases, making it a potential target for drug discovery. However, to date, the successful high-yield purification NOD1 protein has not been reported. Here we describe the large-scale expression of recombinant NOD1 protein in non-adherent mammalian cells. One-step immunoaffinity purification was carried out, yielding highly pure protein with excellent yields. Gel-sieve chromatography studies showed that the purified NOD1 protein eluted almost exclusively as a monomer. Addition of the NOD1 ligand (γ-Tri-DAP) stimulated NOD1 protein oligomerization. Using purified NOD1 protein for nucleotide binding studies by the Fluorescence Polarization Assay (FPA) method, we determined that NOD1 binds preferentially to ATP over ADP and AMP or dATP. We also documented that purified NOD1 protein binds directly to purified pro-apoptotic protein Bid, thus extending recent data that have identified Bid as an enhancer of NOD1 signaling. This expression and purification strategy will enable a wide variety of biochemical studies of mechanisms of NOD1 regulation, as well as laying a foundation for future attempts at drug discovery.     

Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain (LC; 50 kDa) and a binding-translocating heavy chain (HC; 100 kDa) linked through a disulfide bond. The complete sequence of BoNT/A consists of 1296 amino acid residues. The beta-trefoil domain for BoNT/A to which gangliosides bind starts at Ser 1092 and this fragment represents the C-half of the C-terminus of the heavy chain (C-quarter HC or HCQ). The recombinant HCQ DNA was successfully cloned into an expression vector (pET15b), which was used to transform Escherichia coli strain BL21-Star (DE3) for expression. Expression of HCQ was obtained by an extended post-induction time of 15 h at 30 degrees C. The recombinant histidine tagged HCQ protein was isolated and purified by nickel affinity gel column chromatography and its molecular weight was verified by gel electrophoresis. The HCQ was positively identified by antibodies raised against BoNT/A employing immunological dot-blot and Western blot assays. HCQ was shown to bind with synaptotagmin (a known BoNT/A receptor) and gangliosides, indicating that the expressed and purified HCQ protein retains a functionally active conformation.     

A new scaleable method for the purification of botulinum neurotoxin type E

Botulinum neurotoxins belong to the most toxic substances in nature. Well-known as a food poisoning agent for almost two centuries, the beneficial aspects of this bacterial metabolite were rediscovered about 30 years ago. These toxins, which are produced by the anaerobic bacterium Clostridium botulinum are nowadays used to treat a variety of neuro-muscular disorders. The increased demand requires techniques for the production and purification of these toxins on an industrial scale. The method described herein is based on filtration and chromatography procedures only. Precipitation, centrifugation and dialysis steps were consequently excluded to develop a protocol, which can easily be scaled up from the laboratory purification to industrial needs. About 4 mg of BoNT/E were purified from a 10-L batch culture corresponding to an overall recovery of approximately 14%.     

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.     

Production and purification of Clostridium botulinum type C and D neurotoxin

Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay.     

Affinity chromatography purification of type A botulinum neurotoxin from crystalline toxic complex.

Type A botulinum neurotoxin was purified from toxic crystals by adsorption to p-aminophenyl-beta-D-thiogalactopyranoside coupled to CH-Sepharose 4B. At pH 6.3, the toxic complex was held by the binding between the ligand and the hemagglutinin of the complex; the toxin is eluted selectively by dissociating the complex with buffer-saline of pH 7.9. The single-step affinity chromatography recovered 50 to 60% of applied toxicity as preparations of greater than 99% purity.     

Expression and purification of receptor for activated C-kinase 1 (RACK1)

Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large soluble aggregates and thereby lead to false conclusions about the biological activity of RACK1. We developed a purification procedure that gave soluble monodisperse molecules of the protein. The RACK1 gene was cloned and expressed in a pMAL vector. After purification of the resulting MBP-RACK1 fusion protein, RACK1 was excised from MBP by thrombin, rendering RACK1 in a soluble monodisperse form as monitored by fluorimetric static light scattering, gel filtration, and ultracentrifugation. Circular dichroism analysis revealed that RACK1 was properly folded with a T(m) of approximately 62 degrees C and contained the predicted portions of secondary structures. The biological activity of the purified protein was verified by binding to activated protein kinase C. The production of soluble, high-purity RACK1 will allow structural studies and functional in vitro studies to identify interacting partners to this important scaffold protein.     

全长SOD2重组蛋白的可溶表达、纯化、稳定性与跨膜效应

超氧化物歧化酶(SOD)家族是保护细胞免受正常代谢过程中产生的活性氧(ROS)毒性所必需的,含Mn2+离子的超氧化物歧化酶(Mn-SOD,SOD2)是其中最重要的一种。本研究合成了人源SOD2全基因序列,并将其插入带有GST的原核表达载体p GEX-4T-1中,成功构建了GST-SOD2融合蛋白表达质粒。然后,将重组质粒p GEX-4T-1-SOD2转化大肠杆菌BL21(DE3),用IPTG在25℃下诱导表达融合蛋白,得到可溶性GST-SOD2融合蛋白,经GST亲和树脂纯化得到比活为1 788 U/mg的纯蛋白,分子量约为46 k Da。利用凝血酶切去GST标签后经肝素亲和柱纯化得到了电泳纯的SOD2重组蛋白,该蛋白分子量约为25 k Da,与SOD2全长序列的理论分子量相符,比活为2 000 U/mg。两种重组SOD2蛋白在生理条件下都具有良好的SOD活性,且都具有显著的跨膜能力(P0.05)。这些工作为深入研究两种全长重组SOD2蛋白的结构与生物效应建立了基础。     

The protein truncation caused by fusion of PEP-1 peptide and protective roles of transduced PEP-1-MsrA in skin cells

PEP-1 peptide has been used for transduction of native protein into mammalian cells. This work describes the findings that the fusion of PEP-1 to target proteins led to protein truncation likely in a non-protein-specific manner. Approximately 75% of PEP-1-MsrA fusion protein was truncated in the N-terminal region of MsrA between Lys-27 and Val-28 during expression in Escherichia coli and purification. This large protein truncation was also observed in another PEP-1 fused protein, PEP-1- MsrB2, in the N-terminal region of MsrB2. The full-length PEP-1-MsrA protein was rapidly transduced into keratinocyte cells within 15 min. The transduced PEP-1-MsrA was functionally active and could protect skin cells against oxidative stress-and ultraviolet radiation-induced cell death. Collectively, our data demonstrated the protective roles of MsrA in skin cells and, moreover, may raise a concern of protein truncation caused by fusion of PEP-1 about the general use of this peptide for protein transduction.     

Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.     

Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.     

Expression and purification of recombinant poly(A)-specific ribonuclease (PARN)

PARN is a poly(A)-specific ribonuclease that degrades the poly(A) tail of mRNA. We have established conditions for expressing soluble recombinant human PARN. We investigated different Escherichia coli strains, expression vectors, media and growth conditions. We found that PARN expressed from pET33 in BL21(DE3) grown in TB and induced at OD595 approximately 1 with 1 mM IPTG yielded mg amounts of soluble PARN per litre culture. Further, a purification protocol was established to purify PARN. We use His-tag affinity chromatography, HiTrap Q HP ion exchange chromatography and 7-Me-GTP-Sepharose affinity chromatography. This purification procedure render a 90-95% pure PARN. Purified recombinant PARN has enzymatic activity and will be used for further mechanistic and structural studies.     

Expression, purification, and characterization of human type II arginase

Human type II arginase, which is extrahepatic and mitochondrial in location, catalyzes the hydrolysis of arginine to form ornithine and urea. While type I arginases function in the net production of urea for excretion of excess nitrogen, type II arginases are believed to function primarily in the net production of ornithine, a precursor of polyamines, glutamate, and proline. Type II arginases may also regulate nitric oxide biosynthesis by modulating arginine availability for nitric oxide synthase. Recombinant human type II arginase was expressed in Escherichia coli and purified to apparent homogeneity. The Km of arginine for type II arginase is approximately 4.8 mM at physiological pH. Type II arginase exists primarily as a trimer, although higher order oligomers were observed. Borate is a noncompetitive inhibitor of the enzyme, with a Kis of 0.32 mM and a Kii of 0.3 mM. Ornithine, a product of the reaction catalyzed by arginase and a potent inhibitor of type I arginase, is a poor inhibitor of the type II isozyme. The findings presented here indicate that isozyme-selectivity exists between type I and type II arginases for binding of substrate and products, as well as inhibitors. Therefore, inhibitors with greater isozyme-selectivity for type II arginase may be identified and utilized for the therapeutic treatment of smooth muscle disorders, such as erectile dysfunction.     

Expression and purification of Plasmodium falciparum MSP-1(42): A malaria vaccine candidate

The C-terminal 42.10(3) Da portion of the merozoite surface protein (MSP-1) of the human malaria parasite Plasmodium falciparum is of interest, not only because it may constitute an essential part of a future anti-malaria vaccine, but also due to its role during the infection of erythrocytes by the parasite. We have cloned and expressed two synthetic DNA sequences encoding the two prototypic MSP-1(42) variants in E. coli. When over-produced, both proteins form insoluble aggregates which were isolated in high purity and yield. After solubilisation and refolding in vitro, both proteins were purified to homogeneity by a three-step procedure applying Ni-chelate, size exclusion and immuno-affinity chromatography. After purification, both proteins meet key criteria of preparations for clinical use. First, conformational studies suggest proper folding of the proteins, particularly in the region containing two EGF-like domains. Polyclonal serum raised against E. coli produced MSP-1(42) recognizes native MSP-1 in Plasmodium infected erythrocytes as shown by immunofluorescence.     

Expression, purification, and functional testing of recombinant CYR61/CCN1

The human cysteine-rich protein 61 (CYR61/CCN1) belongs to the CCN family of genes which plays an important role in cellular processes such as proliferation, migration, adhesion, and differentiation. These extracellular matrix signaling molecules consist of a modular structure and contain 38 conserved cysteine residues. Previously, we have shown that CYR61 is expressed in human osteoblasts and is regulated by bone-relevant growth factors. The protein also plays a role in angiogenesis. The open reading frame was cloned into a baculovirus expression vector and transfected into SF-21 insect cells. Recombinant protein was expressed as a fusion protein with the Fc-domain of human IgG and purified using affinity chromatography on protein G-Sepharose columns. The chorioallantoic membrane assay verified that blood vessel formation was stimulated by rCYR61. Additionally, human primary mesenchymal stem cells, osteoblasts, and endothelial cells responded to CYR61 treatment by a markedly stimulated proliferation. rCYR61-Fc represents a tool to elucidate its role in cells of the bone microenvironment.     

Expression,purification, and crystal structure of N-terminal domains of human ubiquitin-activating enzyme (E1)

Ubiquitin-activating enzyme (E1) is a key regulator in protein ubiquitination, which lies on the upstream of the ubiquitin-related pathways and determines the activation of the downstream enzyme cascade. Thus far, no structural information about the human ubiquitin-activating enzyme has been reported. We expressed and purified the N-terminal domains of human E1 and determined their crystal structures, which contain inactive adenylation domain (IAD) and the first catalytic cysteine half-domain (FCCH). This study presents the crystal structure of human E1 fragment for the first time. The main structure of both IAD and FCCH superimposed well with their corresponding domains in yeast Uba1, but their relative positions vary significantly. This work provides new structural insights in understanding the mechanisms of ubiquitin activation in humans.