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.     

Nonidentity of chromogranin a and dopamine β-monooxygenase

Dopamine β-monooxygenase (EC 1.14.17.1) and chromogranin A from bovine adrenal chromaffin granules were purified by established procedures and examined for evidence of structural identity. The minimum molecular weights were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to be 74,000 and 35,000, respectively. Amino acid analyses of the two proteins are distinct. Dopamine β-monooxygenase does not possess a free amino terminus, whereas chromogranin A has a leucine amino terminus. Analysis in the protein sequencer showed that chromogranin A contains only a single degradable polypeptide chain. Radioactive S-carboxymethyl derivatives of the two proteins were prepared to compare the soluble peptides after thermolysin digestion. These thermolytic peptides were isolated from columns of Dowex 50-X8 resin and both the peptide and radioactive traces revealed no evidence for similarity of the two proteins, either in toto or in part. Thus, although dopamine β-monooxygenase and chromogranin A may sometimes be copurified, they are distinct entities.     

Complexes of chromogranin A and dopamine beta-hydroxylase among the chromogranins of the bovine adrenal medulla

1. The core proteins of chromaffin granules have been examined by polyacrylamide gel electrophoresis and crossed immunoelectrophoresis against monospecific antisera. 2. Dopamine beta-hydroxylase (dopamine beta-monooxygenase, EC 1.14.17.1) appeared as the major immunogen of the core proteins and accounted for 4 and 8% by weight of the crude lysate and membrane-containing fractions, respectively. 3. The non-ionic detergent, Berol, solubilized dopamine beta-hydroxylase from the membranes in a form which was immunologically identical but of lower relative mobility by crossed immunoelectrophoresis. In the absence of detergent a difference in relative mobility was also noted between the purified enzyme and that contaminated by chromogranin A. These observations suggest that several molecular forms of dopamine beta-hydroxylase may occur which differ in size and/or charge due to interactions with the contaminants under the experimental conditions. 4. The main chromogranin in the crude lysate was absent from electropherograms of the acidic chromogranins (95--96% of total protein in lysate). These were obtained free of dopamine beta-hydroxylase by concanavalin A adsorption at high ionic strength or by acidification in 2 M acetic acid. The main band reappeared upon recombination with dopamine beta-hydroxylase, indicating the presence of some dopamine beta-hydroxylase, possibly as dimers, in this main, chromogranin A band. A protein concentration-dependent aggregate of dopamine beta-hydroxylase-free chromogranin A was detected, with a relative mobility slightly faster than the main band of the crude lysate.     

Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.     

Multiple epitope tagging of expressed proteins for enhanced detection

Three FLAG epitopes have been incorporated into the mammalian expression vector pCMV-5 to create a transient expression vector, p3XFLAG-CMV-7. The vector was designed to express FLAG fusion proteins that can be detected at tenfold lower expression levels than the current FLAG fusion protein expression system. The usefulness of this expression and detection system was demonstrated by expression of bacterial alkaline phosphatase in COS-7 cells. In addition, 3XFLAG bacterial alkaline phosphatase was expressed in Escherichia coli, purified on anti-FLAG M2 affinity gel, and detection of 500 pg of purified protein by Western blot analysis is demonstrated.     

Chromogranin from normal human adrenal glands: purification by monoclonal antibody affinity chromatography and partial N-terminal amino acid sequence

A method is presented for the purification of human chromogranin from adrenal glands obtained at autopsy. The procedure involved homogenization of whole glands in aqueous buffer, salt precipitation, affinity chromatography using a highly specific monoclonal antibody (LK2H10) and reverse-phase high-pressure liquid chromatography. Chromogranin purified from autopsy adrenal glands revealed a high degree of polypeptide heterogeneity when analyzed by silver-stained SDS polyacrylamide gels. Greater than 90% of the protein was represented by a cluster of polypeptides with an Mr = 70 000 (i.e. chromogranin A), while the remaining protein was highly disperse in molecular weight. That these various polypeptides were in fact chromogranin was shown by Western blotting using monoclonal antibody LK2H10. About 6 nmol of chromogranin were obtained from 97 g of starting adrenals which was estimated to be a 25% yield and a 250-fold enrichment from adrenal homogenates. Critical to achieving reasonable yields of this protein was the need for particular low pH buffers for resuspension of chromogranin after solvent removal steps. Chromogranin purified from human adrenal glands was similar in amino acid composition, and identical in the N-terminal amino acid sequence (24 residues) to bovine chromogranin A. A secondary sequence representing 25% of the total protein and missing the first three residues of the N-terminus suggested the possibility of N-terminal processing of chromogranin in situ. The conservation of the N-terminal amino acid sequence of human and bovine chromogranin contrasts with the strong sequence variability predicted by antisera cross-reactivity and suggests that the N-terminus of chromogranin may be critical for its biological activity.     

Heterologous expression, purification and characterization of heterodimeric monellin

Monellin is an intensely sweet-tasting protein present in the berry of Dioscoreophyllum cumminsii. Naturally occurring monellin (double chain monellin) is a heterodimer of two subunits commonly referred to as chain A and chain B. Monellin is a good model system for structural and dynamic studies of proteins. Single chain monellin has been generated by covalently linking the two subunits of naturally occurring double chain monellin, and has been used extensively for folding and unfolding studies, as well as for protein aggregation studies. There are, however, relatively few reports on such studies with double chain monellin. The primary difficulty associated with studies using double chain monellin appears to be the lack of a standard purification method. Here, a simple method for the purification of double chain monellin is presented. The genes encoding the two chains of monellin were cloned into a modified pETDUET vector under separate T7 promoters. The expression vector containing the genes of the two chains was expressed in E. coli BL21 Star (DE3). The expressed protein was purified using two steps of chromatography, ion exchange chromatography and gel filtration chromatography. This expression system consistently produced 40 mg of pure double chain monellin per litre of E. coli culture, in the correctly folded native state. The purity of the protein was confirmed by mass spectrometry and SDS-PAGE analysis. The purified protein was characterized using different spectroscopic methods, and the spectra obtained were in good agreement with the published spectra of naturally occurring double chain monellin.     

应用细菌鞭毛递呈的随机肽库研究丙型肝炎病毒核心蛋白B细胞抗原表位

设计了利用大扬杆菌鞭毛蛋白递呈的随机十二肽库研究ＨＣＶ核心蛋白Ｂ细胞抗原位的实验程序：１利用大肠杆有达质粒ｐＱＥ－３０有达并纯化ＨＣＶ核心蛋白Ｐ１９;２利用Ｐ１９蛋白亲和层析纯化ＨＣＶ感染者血清的抗ＨＣＶ核心蛋白多克隆抗体;     

Studies on Limulus amoebocyte lysate. III. Purification of an endotoxin-binding protein from Limulus amoebocyte membranes

A protein that has been isolated from Limulus polyphemus amoebocyte membranes binds endotoxin. The protein was purified by two independent methods, organic solvent extraction and affinity chromatography, both followed by gel filtration. Immunologic studies confirm that the protein is a component of amoebocyte membranes. Although without enzymatic activity, the binding protein enhances Limulus lysate gelation. As a membrane-associated endotoxin binding "protein," it may be involved in Limulus lysate coagulation, which is initiated by minute amounts of Gram-negative bacterial endotoxin. The protein has an apparent molecular weight of 80,000.     

Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene, which expresses a novel collagenase activity.

In order to examine the potential role of bacterial collagenases in periodontal tissue destruction, we recently isolated a gene, prtC, from Porphyromonas gingivalis ATCC 53977, which expressed collagenase activity (N. Takahashi, T. Kato, and H. K. Kuramitsu, FEMS Microbiol. Lett. 84:135-138, 1991). The nucleotide sequence of the gene has been determined, and the deduced amino acid sequence corresponds to a basic protein of 37.8 kDa. In addition, Southern blot analysis indicated that the prtC gene is conserved among the three major serotypes of P. gingivalis. The enzyme has been purified to near homogeneity from Escherichia coli clone NTS1 following Mono Q anion exchange and sequential gel filtration chromatography. The molecular mass of the purified enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be ca. 35 kDa, and the active enzyme behaved as a dimer following gel filtration chromatography. The collagenase degraded soluble and reconstituted fibrillar type I collagen, heat-denatured type I collagen, and azocoll but not gelatin or the synthetic collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg. Enzyme activity was enhanced by Ca2+ and inhibited by EDTA, sulfhydryl-blocking agents, and the salivary peptide histatin. Preliminary evidence for the existence of a second collagenase expressed by strain 53977 was also obtained.     

Purification of Porcine Phospholamban Expressed inEscherichia coli

Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathioneS-transferase has been expressed in JM109Escherichia colicells. The expressed fusion protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS–gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3–4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.     

Purification and characterization of dopamine beta-hydroxylase from bovine adrenal medulla.

A new purification procedure that permits large-scale purification of dopamine beta-hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1% Triton X-100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE-cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis. The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2-terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2-terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2-terminal amino acids do not support the suggestion that dopamine beta-hydroxylase and chromogranin A contain identical peptide chains.     

Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.

Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.     

Characterization of LC-HCC fusion protein of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- H(CC) in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.     

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E. coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding approximately 42 and 36mg EPF from 300ml bacterial and 1L Sf9 cultures, respectively. The preparations were highly purified (#10878;99% purity on SDS-PAGE for the bacterial products and #10878;97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immunosuppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity.     

Purification and partial characterization of cellular retinoic acid-binding protein from human placenta

Cellular retinoic acid-binding protein (CRABP) has been purified to homogeneity from human placenta by a series of procedures, including acetone powder extraction, gel filtration on Sephadex G-50, and ion-exchange chromatography on DEAE-cellulose and on SP-Sephadex. Cellular retinol-binding protein (CRBP) was isolated concurrently. CRABP was purified 75,400-fold, based on total soluble acetone powder extract of placenta. The protein is a single polypeptide chain with a molecular mass of 14,600 Da, estimated by sodium dodecyl sulfate (SDS) gel electrophoresis or gel filtration, and has an isoelectric point of 4.78 (apo-CRABP, 4.82). On analysis of absorption and fluorescence spectra, the protein was seen to exhibit an absorption peak at 350 nm, fluorescence excitation maxima at 350 and 370 nm, and a fluorescence emission maximum at 475 nm. Human CRABP was immunologically distinct from human CRBP and serum retinol-binding protein.     

Isolation and characterization of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system from Bacillus megaterium has been resolved into three components, 1) a NADPH-specific, FMN-containing flavoprotein reductase, molecular weight 55-60 000; 2) an iron-sulfur protein, molecular weight 13,000 and 3) cytochrome P-450meg, molecular weight 52,000. The cytochrome component has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, and its amino acid composition has been determined. Cytochrome P-450meg has a pI of 4.9, a Stokes radius of 27 A and a sedimentation constant of 3.3 S. Electron paramagnetic resonance and optical spectra are typical of a low-spin cytochrome P-450. The fluorescence spectrum is indicative of a tryptophane residue in a relatively non-polar environment. In recombination experiments, the electron flow was shown to proceed from the reductase via the iron-sulfur protein to the cytochrome. It is also possible to exchange the different components of the mitochondrial 11 beta-hydroxylase system from bovine adrenals for corresponding components in B. megaterium. Substrate specificity studies indicate that only steroids with a 3-oxo-delta 4-configuration are hydroxylated by the B. megaterium hydroxylase system. When oxidizing agents were used, hydroxylation occurred both in positions 15 alpha and 15 beta. Further substrate specificity studies have shown that aniline and imipramine can function as substrates for the bacterial system.     

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.     

beta-Granins: 21 kDa co-secreted peptides of the insulin granule closely related to adrenal medullary chromogranin A

Three closely related forms of a 21 kDa protein which is co-secreted with insulin have been purified and analysed. These differed in behaviour on ion-exchange chromatography but were indistinguishable by their susceptibility to staphylococcal V8 proteinase digestion, amino acid composition or N-terminal amino acid sequence. Their amino acid composition and N-terminal sequences were remarkably similar to adrenal medullary chromogranin A, a much larger protein (72 kDa). Antibodies to chromogranin A also reacted strongly with the 21 kDa protein in isolated insulin granules. It is concluded that the 21 kDa proteins either represent a repeated domain within the chromogranin molecule or a closely related gene product. The name beta-granin is proposed for these proteins.