N-terminal Histidine Tag Research Articles

Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oligomerization

Streptolysin O belongs to the class of thiol-activated toxins, which are single chain, four-domain proteins that bind to membranes containing cholesterol and then assemble to form large oligomeric pores. Membrane binding involves a conserved tryptophan-rich sequence motif located within the C-terminally located domain 4. In contrast, sites involved in oligomerization and pore formation have been assigned to domains 1 and 3, respectively. We here examined the functional properties of domain 4, which was recombinantly expressed with an N-terminal histidine tag for purification and an additional cysteine residue for covalent labeling. The fluorescently labeled fragment readily bound to membranes, but it did not form oligomers nor lyse cell membranes. Moreover, the labeled fragment did not detectably become incorporated into hybrid oligomers when combined with lytically active full-length toxin. However, when present in large excess over the active toxin, the domain 4 fragment effected reduction of hemolytic activity and of functional pore size, which indicates interference with oligomerization of the lytically active species. Our findings support the notion that domain 4 of the streptolysin O molecule may fold autonomously, is essential for membrane binding and is capable not of irreversible but of reversible association with the entire toxin molecule.

Purification and Functional Analysis of the Copper ATPase CopA of Enterococcus hirae

The Enterococcus hirae ATPase CopA is a member of the recently discovered heavy metal ATPases and shares 43% sequence identity with the human Menkes and Wilson copper ATPases. To study CopA biochemically, it was overexpressed in E. coli with an N-terminal histidine tag and purified to homogeneity by nickel affinity chromatography. The purified CopA catalyzed ATP hydrolysis with a Vmax of 0.15 μmol/min/mg and a Km for ATP of 0.2 mM and had an optimum pH of 6.25. The activity was 3- to 4-fold stimulated by reconstitution into proteoliposomes. The enzyme formed an acylphosphate intermediate. Its kinetics of formation and the effects of inhibitors and metal ions upon it support a function of CopA in copper transport. Purification and functional reconstitution of CopA provides the basis to study copper transport in vitro.

Cloning, purification and characterisation of Staphylococcus warneri lipase 2

A gene encoding an extracellular lipase was identified in Staphylococcus warneri 863. The deduced lipase is organised as a prepro-protein and has significant similarity to other staphylococcal lipases. The mature part of the lipase was expressed with an N-terminal histidine tag in Escherichia coli, purified and biochemically characterised. The results show that the purified lipase (named SWL2) combines the properties of the staphylococcal lipases characterised so far. It has both a high preference for short chain substrates and surprisingly, it also displays phospholipase activity. Homology alignment was used to analyse sequence–function relationships of the staphylococcal lipase family with the aim to identify the structural basis underlying the different properties of the staphylococcal lipases.

Aphid Acquisition and Cellular Transport of Potato leafroll virus-like Particles Lacking P5 Readthrough Protein

ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric virus-like particles (VLP) when infected with a recombinant baculovirus Ac61 that contained the Potato leafroll virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculovirus that expressed cDNA copies of the PLRV genome RNA, did not produce virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of virus through aphid vectors.

Expression and purification of histidine-tagged proteins from the gram-positive Streptococcus gordonii SPEX system.

Streptococcus gordonii (S. gordonii) has been used as a gram-positive bacterial expression vector for secreted or surface-anchored recombinant proteins. Fusion of the gram-positive bacterial N-terminal signal sequence to the target protein is all that is required for efficient export. This system is termed SPEX for Surface Protein EXpression and has been used to express proteins for a variety of uses. In this study, the SPEX system has been further developed by the construction of vectors that express polyhistidine-tagged fusion proteins. SPEX vectors were constructed with an N-terminal or C-terminal histidine tag. The C-repeat region (CRR) from Streptococcus pyogenes M6 protein and the Staphylococcus aureus nuclease A (NucA) enzyme were tested for expression. The fusion proteins were purified using metal affinity chromatography (MAC). Results show that the fusion proteins were expressed and secreted from S. gordonii with the His tag at either the N- or C-terminal position and could be purified using MAC. The M6 fusions retained immunoreactivity after expression and purification as determined by immunoblots and ELISA analyses. In addition, NucA fusions retained functional activity after MAC purification. The M6-His and NucA-His fusions were purified approximately 15- and 10-fold respectively with approximately 30% recovery of protein using MAC. This study shows that the polyhistidine tag in either the N- or C-terminal position is a viable way to purify secreted heterologous proteins from the supernatant of recombinant S. gordonii cultures. This study further illustrates the value of the SPEX system for secreted expression and purification of proteins.

Sequestration of the Active Site by Interdomain Shifting: CRYSTALLOGRAPHIC AND SPECTROSCOPIC EVIDENCE FOR DISTINCT CONFORMATIONS OF l-3-HYDROXYACYL-CoA DEHYDROGENASE

Sequestration of the Active Site by Interdomain Shifting: CRYSTALLOGRAPHIC AND SPECTROSCOPIC EVIDENCE FOR DISTINCT CONFORMATIONS OF l-3-HYDROXYACYL-CoA DEHYDROGENASE

PhrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase.

A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechocuccus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.

Rhodopsin kinase: Expression in mammalian cells and a two-step purification

A suitable system for expression of the rhodopsin kinase (RK) gene and its mutants is needed for structure-function studies of RK. Previously, investigation of the baculovirus system showed satisfactory production of RK, but posttranslational isoprenylation was deficient. We now report on a comparative study of expression of the RK gene in yeast (Pichia pastoris), COS-1 cells and in an HEK293 stable cell line. Expression in COS-1 cells, by using pCMV5 vector, is the most satisfactory. A two-step procedure for purification of the expressed enzyme with an N-terminal histidine tag has been developed. The purified enzyme has correct posttranslational modifications and shows a somewhat broader pH vs. catalytic activity profile than the wild-type enzyme.

New fusion protein systems designed to give soluble expression in Escherichia coli

Three native E. coli proteins—NusA, GrpE, and bacterioferritin (BFR)—were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37°C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37°C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-γ were also expressed in E. coli at 37°C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 382–388, 1999.

New fusion protein systems designed to give soluble expression inEscherichia coli

Three native E. coli proteins-NusA, GrpE, and bacterioferritin (BFR)-were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37 degrees C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37 degrees C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-gamma were also expressed in E. coli at 37 degrees C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography.

Low-salt crystallization of T7 RNA polymerase: a first step towards the transcription bubble complex.

DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. This process, which decodes the genetic information from DNA, is one of the most significant events in a biological system. The crystallization of both native and a chimeric T7/T3 RNAP using high salt conditions has been reported previously but these conditions proved unsuitable for DNA-RNAP complex formation since at high salt concentrations the DNA binding affinity to RNAP is reduced. A search for low-salt crystallization conditions has yielded new low-salt crystals of native T7-RNAP, a chimeric T7-RNAP (T7/T3 RNAP) which contains the T3 promoter recognition sequence, and a T7-RNAP containing an N-terminal histidine tag. The crystals, which are better suited for DNA-RNAP complex formation, belong to space group P3121 with a = 136, c = 156 A, contain a single molecule per asymmetric unit and diffract to 2.7 A resolution. Packing analysis shows that the new low-salt crystals have packing contacts similar to those observed in the high-salt T7-RNAP crystals reported previously. The diffraction anisotropicity observed in crystals of T7 RNAP is explained in term of crystal packing.

C3a(desArg) does not bind to and signal through the human C3a receptor

Contradictory results have been published in the past regarding the functional responses of different cell types to the anaphylatoxin C3a and its natural catabolite C3a(desArg). To elucidate the interaction of the C3a receptor (C3aR) with its ligand(s) we studied the binding of human recombinant C3a (rC3a) and rC3a(desArg) to RBL-2H3 transfectants which express the C3aR. As the addition of 11 aminoterminal amino acids did not alter the functional activity of the recombinant C3a as compared to serum-derived C3a the specific binding of rC3a and rC3a(desArg) to the transfectants could be determined by flow cytometry using a monoclonal antibody (mab) against their N-terminal histidine tag. Recombinant C3a bound to the C3aR with a half maximal concentration of about 3 nM whereas no evidence for a binding of rC3a(desArg) could be obtained. Furthermore, rC3a(desArg) did not signal through the C3aR. Neither the release of lysosomal N-acetyl-β- d-glucosaminidase nor the directional migration of C3aR-expressing RBL-2H3 transfectants could be detected in response to rC3a(desArg) whereas rC3a was highly active in both assays. Our data demonstrate a defined ligand specificity of the C3aR for the anaphylatoxin C3a. Its natural catabolite C3a(desArg), however, does not signal through the C3aR. Modulating effects of C3a(desArg) on the synthesis of cytokines in human monocytes and B lymphocytes may therefore be induced by receptor-independent mechanisms while their in vivo relevance remains as yet undefined.

Heterologous expression and characterization of endoglucanase I (EGI) from Trichoderma viride HK-75.

Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique transglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of egl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino acids that has extensive similarity (93.8%) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredoxin and C-terminal histidine tag) led to a large production of a nonglycosylated protein of 62.5 kDa. However, it formed an insoluble inclusion body. Upon denaturation with 8 M urea followed by dialysis and successive purification, the enzymatically active recombinant EGI (rEGI) was obtained at a level as high as 18.3 mg/l of 1,000 ml of culture. The rEGI had 67.8% activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The optimum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5 degrees C, respectively. The rEGI also had narrower CMCase ranges than nEGI in pH and temperature stabilities. However, the catalytic and transglycosylation abilities against cellotriose of rEGI were comparable to those of nEGI. These results suggest that the glycosylation is important for the stabilities of EGI but not critical for the essential enzymatic capacity.

Antitumor Response Elicited by a Superantigen- Transmembrane Sequence Fusion Protein Anchored onto Tumor Cells

Superantigens stimulate T cells bearing certain TCR beta-chain variable regions when bound to MHC II molecules. We investigated whether the superantigen toxic shock syndrome toxin-1 (TSST1) could induce an antitumor immune response when anchored onto MHC II-negative tumor cells. Our approach was to facilitate association of TSST1 with cell membranes by fusing its coding region to the transmembrane region (TM) sequence of the proto-oncogene c-erb-B-2. TSST1-TM was expressed in bacteria with an N-terminal histidine tag and purified using nickel-agarose affinity chromatography. Purified TSST1-TM added to cultures of several different MHC II-negative tumor cells spontaneously associated with cell membranes, as detected by flow cytometry. Because superantigens can direct cell-mediated cytotoxicity against MHC II-positive cells, a TM fusion protein lacking the TSST1 MHC II binding domain (TSST(88-194)-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST(88-194)-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST(88-194)-TM-coated tumor cells mounted a systemic response that resulted in significant antitumor immunity as measured by regression of a parental tumor challenge. TSST1-TM and TSST(88-194)-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation.

The lactose transporter of Staphylococcus aureus--overexpression, purification and characterization of the histidine-tagged domains IIC and IIB.

The lactose-specific enzyme II (IICBlac) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) of Staphylococcus aureus couples translocation to phosphorylation of the transported lactose. It is composed of the N-terminal membrane-bound IIC domain, which includes the sugar-binding site, and the C-terminal IIB domain, which contains the phosphorylation site at Cys476. IIC (residues 1-461) fused with a C-terminal affinity tag of six histidine residues and IIB (residues 461-570) fused with an N-terminal histidine tag were overexpressed in Escherichia coli and purified by Ni2+ chelate affinity chromatography. 2 mg of IIClac-His6 obtained from 10 g of cells and 12 mg of His6-IIBlac obtained from 8 g of wet cells were purified to homogeneity. 56% of the total IIClac-His6 activity present in the membranes could be recovered. Purification by affinity chromatography yields the opportunity to exchange the detergent. The Km determined in an activity assay for IIClac-His6 in the presence of the histidine-tagged IIBlac domain (His6-IIBlac) was similar to the Km determined for histidine-tagged IICBlac-His [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798-804], suggesting that substrate affinity is barely influenced by the expression of the domains as separate proteins. The Vmax is reduced by a factor of 25 compared with IICBlac-His. His6-IIBlac also complements the activity of the IICBlac mutant C476S, which possesses an inactive IIB domain. This result indicates that IIC and IIB are flexibly linked in such a way that free His6-IIBlac can displace the inactive IIB domain fron its contact site on the IIC domain. His6-IIBlac is shorter and more stable than a previously constructed IIB domain (IIBlac-His) [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798-804)], which contained a C-terminal histidine tag. The Km values for phosphoenolpyruvate-dependent phosphorylation of His6-IIBlac and IIBlac-His are nearly indistinguishable, suggesting that the location of the affinity tag either at the N-terminal or at the C-terminal end of the domain does not influence the substrate affinity.

Expression and Rapid Purification of an Aedes aegypti Salivary Allergen by a Baculovirus System

Mosquito salivary proteins cause allergic reactions in humans. Recombinant salivary allergens will facilitate both the diagnosis and immunotherapy of mosquito allergy. The Aed a 1, a 68-kD apyrase in the saliva of Aedes aegypti, has been demonstrated to be an allergen which binds to the IgE of mosquito-allergic subjects. The baculovirus expression vector pBlueBacHis C equipped with an N-terminal histidine tag was used to express the Aed a 1 protein. The cDNA coding for the 3′ significant portion of Aed a 1 (Aed a 1 3′) (150–562 amino acid residues) was cloned into pBlueBacHis C. The rAed a 1 3′ protein expressed by recombinant baculovirus was verified by immunoblot using anti-Aed a 1 and anti-histidine antibodies, respectively. The histidine-tagged fusion protein was purified to apparent homogeneity from infected Sf 9 cells by Ni²⁺ resin affnity chromatography. Both immunoblot and ELISA showed that the purified rAed a 1 3′ binds to the IgE and IgG in mosquito allergic sera, indicating that the antigenicity of the rAed a 1 3′ is identical to the native Aed a 1 of Aedes aegypti saliva.

Cloning and Expression of a Prokaryotic Enzyme, Arginine Deiminase, from a Primitive Eukaryote Giardia intestinalis

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Overexpression and Characterization of the Human Mitochondrial and Cytosolic Branched-chain Aminotransferases

We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profiles, and the far UV circular dichroism spectra show that no global structural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form. On the other hand, the near UV circular dichroism spectra suggest differences in the local environment surrounding tyrosines within these proteins. Both enzymes require a reducing environment for maximal activity, but the mitochondrial enzyme can be inhibited by nickel ions in the presence of reducing agents, while the cytosolic enzyme is unaffected. Chemical denaturation profiles of the proteins show that there are differences in structural stability. Titration of -SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme. Two -SH groups are titrated in the native form of the mitochondrial enzyme, leading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity. Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.

A Novel Assay Reveals a Role for SolubleN-Ethylmaleimide-sensitive Fusion Attachment Protein in Mannose 6-Phosphate Receptor Transport from Endosomes to the Trans Golgi Network

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.

The effects of truncations of the small subunit on m-calpain activity and heterodimer formation.

In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a delta 86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-delta 86, -delta 99, -delta 107, and -delta 116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-delta 125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from delta 86, leaving residues 87-245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245-270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys103-->Ser-80k/delta 86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca(2+)-induced dissociation is involved in calpain activation.