Authentic Protein Research Articles

Expression and Purification of HIV-I p15NC Protein inEscherichia coli

An efficient method for the expression and purification of nucleocapsid precursor protein (p15NC) from HIV-I (BH 10 isolate) was developed and used to obtain large quantities of this viral protein for structural studies, protein biochemistry, and high-throughput screening efforts. We have engineered an existing p15NC clone into a new vector developed at the University of Heidelberg, Germany. Using PCR, we introduced new restriction sites and a strong ribosome-binding site in the p15NC gene and expressed authentic p15NC protein. Our protocol enabled us to rapidly obtain soluble and highly stable p15NC expressed inEscherichia coliand to purify several milligrams of p15NC to homogeneity. In the current purification scheme, lysis of cell paste followed by a simple three-step FPLC procedure yields about 0.4–0.5 mg of purified p15NC per gram ofE. colicell paste expressing the protein with an overall yield of 45%. The purified p15NC retained its ability to bind full-length HIV-I p15NC mRNA in solution- or solid-phase-based assays. A specific stem-loop forming RNA fragment (24-mer) and its antisense DNA oligomer (21-mer) derived from the full-length p15NC mRNA were also able to bind to p15NC. In addition, antisense DNA oligos with bulky 5-iodouracil and 5-iodocytidine substituents were able to bind to p15NC with little or no perturbations as assessed by their ability to compete with the full-length p15NC mRNA in filter-binding competition assays. In addition, RNA-dependent cleavage of the purified p15NCin vitroby HIV-I protease occurred at rates similar to those reported previously.

The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes.

Exonucleolytic degradation of the poly(A) tail is often the first step in the decay of eukaryotic mRNAs and is also used to silence certain maternal mRNAs translationally during oocyte maturation and early embryonic development. We previously described the purification of a poly(A)-specific 3'-exoribonuclease (deadenylating nuclease, DAN) from mammalian tissue. Here, the isolation and functional characterization of cDNA clones encoding human DAN is reported. Recombinant DAN overexpressed in Escherichia coli has properties similar to those of the authentic protein. The amino acid sequence of DAN shows homology to the RNase D family of 3'-exonucleases. DAN appears to be localized in both the nucleus and the cytoplasm. It is not stably associated with polysomes or ribosomal subunits. Xenopus oocytes contain nuclear and cytoplasmic DAN isoforms, both of which are closely related to the human DAN. Anti-DAN antibody microinjected into oocytes inhibits default deadenylation during progesterone-induced maturation. Ectopic expression of human DAN in enucleated oocytes rescues maturation-specific deadenylation, indicating that amphibian and mammalian DANs are functionally equivalent.

The defense-related rice gene Pir7b encodes an alpha/beta hydrolase fold protein exhibiting esterase activity towards naphthol AS-esters.

Acquired resistance of rice to Pyricularia oryzae, the causing agent of rice blast, can be induced by inoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have previously cloned a cDNA and a corresponding gene (Pir7b) whose transcripts accumulate upon infiltration with the resistance-inducing bacteria. The putative encoded product Pir7b exhibits significant sequence similarity to two recently cloned hydroxynitrile lyases from Manihot esculenta (cassava) and Hevea brasisliensis, enzymes involved in the release of hydrogen cyanide from cyanogenic glycosides. As rice does not contain cyanogenic glycosides, a similar function of Pir7b appears unplausible. In order to functionally characterize the protein, recombinant Pir7b was produced in Escherichia coli and Saccharomyces cerevisiae. We show that recombinant Pir7b does not have hydroxynitrile lyase activity, but exhibits esterase activity towards naphthol AS-acetate. Using Pir7b-specific antibodies, we show that the protein accumulates in rice leaves inoculated with P. syringae pv. syringae. Both the recombinant and the authentic proteins have an apparent molecular mass of 32 kDa (28.8 kDa calculated) and seem to be active as monomers. Pir7b esterase also exhibits sequence similarity to several expressed sequence tags of Arabidopsis thaliana, indicating that it belongs to a family of proteins widely occuring in plants.

Expression of a Plastidic ATP/ADP Transporter Gene inEscherichia coli Leads to a Functional Adenine Nucleotide Transport System in the Bacterial Cytoplasmic Membrane

Recently, a second type of eucaryotic adenine nucleotide transporter located in the inner envelope membrane of higher plants has been identified at the molecular level (Neuhaus, H. E., Thom, E., Möhlmann, T., Steup, M., and Kampfenkel, K. (1997) Plant J. 11, 73-82). Here we have analyzed the biochemical properties of this ATP/ADP transporter from Arabidopsis thaliana (AATP1, At). This analysis was carried out by expressing a cDNA encoding this carrier as a histidine-tagged chimeric protein heterologously in Escherichia coli. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-induced E. coli cells were able to import radioactively labeled [alpha-32P]ATP. Uninduced E. coli cells did not import [alpha-32P]ATP. Further control experiments revealed that IPTG induction did not promote import of other phosphorylated or unphosphorylated metabolites into the bacterial cell indicating the specificity of [alpha-32P]ATP transport. [alpha-32P]ATP uptake into induced E. coli cells was linear with time for several minutes allowing for determination of kinetic constants. The apparent Km for ATP was 17 microM which is close to values reported on the authentic protein in isolated plastids. ADP was a strong competitive inhibitor of -alpha-32P-ATP uptake (Ki ADP 3.6 microM). Other metabolites like AMP, ADP glucose, UTP, UDP, NAD, and NADP did not influence [alpha-32P]ATP uptake. IPTG-induced E. coli cells preloaded with [alpha-32P]ATP exported radioactively labeled adenylates after exogenous addition of unlabeled ATP or ADP indicating a counter exchange mechanism of transport. The biochemical properties of the heterologously expressed AATP1 gene product demonstrated that the protein is functionally integrated in the cytoplasmic membrane of E. coli. This is the first report of the functional expression of a plant membrane protein in E. coli leading to new transport properties across the cytoplasmic membrane. The functional integration of a plant membrane protein in the cytoplasmic membrane of E. coli offers new possibilities for future studies of the structural and mechanistic properties of this transporter. Since IPTG induction allowed synthesis of a 67-kDa protein in E. coli, which was subsequently specifically enriched by metal-chelate chromatography, this procaryotic heterologous expression system might provide a suitable system for overproduction of membrane proteins of eucaryotic origin in the near future.

17] Purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10

Publisher Summary The chapter presents a study on the purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10. The chapter describes a large-scale purification procedure of S. cerevisiae hsp60 expressed in Pichia pastoris. Yeast cpnl0 was isolated by taking advantage of the fact that bacterial GroEL forms a functional complex with co-chaperonins from mitochondria or chloroplasts. Chaperonin system is not involved in the folding of all mitochondrial proteins. Assaying the interaction of purified hsp60 and cpnl 0 with authentic substrate proteins should give insight into the specificity of the mitochondrial chaperonin system. In addition, a comparison of the bacterial GroEL/ES system with the eukaryotic hsp60/cpnl0 system may help to elucidate the chaperonin reaction cycle. The chapter describes the procedure for purification of recombinant heat shock protein 60, purification of recombinant authentic and hexahistidine-tagged chaperonin 10, and also mentions the characterization of purified yeast chaperonins.

Heterologous expression of Hydrogenobacter thermophilus cytochrome c-552 in the periplasm of Pseudomonas aeruginosa

Thermostable cytochrome c-552 of Hydrogenobacter thermophilus was heterologously expressed in the periplasm of Pseudomonas aeruginosa using a fusion gene coding for the P. aeruginosa cytochrome c-551 signal peptide and the H. thermophilus mature cytochrome c-552. The values of the thermal denaturation parameters of periplasmically expressed cytochrome c-552 were identical to those of the authentic protein purified from H. thermophilus.

Fluorescence Detection of Calcium-Binding Proteins with Quinoline Ca-Indicator Quin2

We have established a fluorescence method to detect calcium-binding proteins by making use of the quinoline Ca indicator quin2. Authentic calcium-binding proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electrophoretically transferred onto polyvinylidene difluoride membranes. Transfers were incubated with nonradioactive calcium ions, then with quin2 to detect the calcium-binding proteins as fluorescent bands by illumination with UV light. Calmodulin and parvalbumin of EF hand conformation calcium-binding type were clearly identified. Quin2 distinguished smooth muscle α-actinin from skeletal muscle α-actinin; the former was faintly fluorescent, having a low affinity for calcium ions. In whole myofibril preparations from skeletal muscles, troponin-C, connectin (titin), and nebulin were intensely fluorescent, being shown to have calcium-binding ability. The fluorescence method is an accurate, safe, and simple procedure to detect the binding of calcium ions to proteins following electrophoresis. The overlay technique described can be completed within 15 h and detects as little as 38 ng/well of troponin-C in the starting sample.

Localization of the amino terminus of the hepatitis B virus core antigen within the core particle

The position of the amino terminus of the hepatitis B virus core antigen (HBcAg) within the core particle was studied. For this purpose, three recombinant analogs of HBcAg were designed. One analog, HBcAgR, was identical in amino acid sequences to the core polypeptide of the hepatitis B virus; the second, HBeAgR, differed from the authentic protein in deletion of 39 carboxy-terminal amino acids. The amino acid sequences of the third polypeptide, HBeΔN and of HBeAgR were similar, HBeΔN differed from HBeAgR only in replacement of 3 N-terminal amino acids by 16 amino acids of β-galactosidase. The HBcAg analogs were compared with respect to their reaction with monoclonal antibody (mAb E1A7) to the amino-terminal linear epitope of hepatitis B virus e antigen. Although able to assemble into virus-like particles, the three analogs of HBcAg, reacted differently with mAb E1A7. It was demonstrated that mAb E1A7 reacted with both native and denatured HBeAgR. HBeΔN was not recognized by mAb E1A7. In contrast, HBcAgR reacted with mAb E1A7 only when denatured. Native HBcAgR did not react with mAb E1A7 when assembled into particles. Thus evidence was obtained that the amino terminus of HBcAg is not exposed on the particle surface.

The in vitro biosynthesis of an authentic protein

The in vitro biosynthesis of an authentic protein

Cloning, sequence analysis and expression of the major outer capsid protein gene of an aquareovirus.

The nucleotide and deduced amino acid sequences of genome segment 10 of aquareovirus strain SBR, encoding the major outer capsid protein (VP7), have been determined. Genome segment 10 of SBR virus is 986 nucleotides long and encodes a polypeptide of 298 amino acids with a predicted molecular mass of 32,430 Da. There are 26 non-translated nucleotides at the 5' end and 66 non-translated nucleotides at the 3' end. Using a recombinant baculovirus system, the VP7 protein of SBR virus was expressed to a high level. The baculovirus-produced VP7 protein was similar both in its size and antigenic properties to the authentic aquareovirus VP7 protein. Antiserum from a rabbit immunized with the baculovirus-produced VP7 protein failed to neutralize the homologous aquareovirus strain. As determined by Western blotting, this antiserum reacted with aquareovirus strains belonging to the same genogroup as SBR virus, but did not react with aquareovirus strains belonging to the other genogroups.

The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Expression of theSolfolobus acidocaldariusRieske Iron Sulfur Protein II (SOXF) with the Correctly Inserted [2FE–2S] Cluster inEscherichia coli

The Rieske protein II (Schmidtet al.,1996,FEBS Lett.388, 43–46) from the thermoacidophilic crenarcheonSulfolobus acidocaldarius(DSM 639) was expressed inE. colicells. The full length protein was strictly bound to theE. colimembranes and could only be removed by detergent treatment indicating the presence of a membrane anchor. The iron sulfur cluster was correctly inserted into a fraction of the full length protein and much more effectively into a soluble form created by the deletion of the 45 N-terminal amino acids. The soluble form of the protein displayed the typical spectroscopic properties of a respiratory Rieske protein. The midpoint potential was +375 mV determined by CD redox potentiometry. The presented data demonstrate that the structure of the recombinant protein is very similar or identical to the authentic protein making this a powerful model system for the studies of Rieske proteins by site directed mutagenesis.

Chloroplast SRP54 Interacts with a Specific Subset of Thylakoid Precursor Proteins

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.

Identification, subcellular localization, and developmental studies of oleosins in the anther of Brassica napus.

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15-residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of approximately 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N-terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of lowdensity organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule-filled plastids and lipid-containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low-density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil-body oleosins share common epitopes at the generally non-conserved C-terminal domain with the sporophytic tapetum oleosins.

Multiplicity of uses of monoclonal antibodies that define papillomavirus linear immunodominant epitopes

During the last 10 yr, we have derived monoclonal antibodies from animals immunized with denatured bovine papillomaviruses type 1 major capsid (L1) protein, mapped their corresponding immunodominant epitopes to within a single amino acid (aa), and compared the reactivity of authentic L1 proteins to the predicted response by collinear analysis of the aa sequences of the same and other papillomaviruses (PVs). The data obtained from this approach has provided us with new insights into the sensitivity and specificity of the antibody response to viral proteins. We have included here some observations and conclusions that appear to be generic for the immune response, some of which might have applications for working with linear epitopes in other experimental systems.

The PGK-TIM fusion protein from Thermotoga maritima and its constituent parts are intrinsically stable and fold independently.

In the hyperthermophilic bacterium Thermotoga maritima, the two glycolytic enzymes phosphoglycerate kinase (PGK) and triosephosphate isomerase (TIM) are covalently connected forming a tetrameric single-chain PGK-TIM fusion protein. A frameshift allows the translation of PGK alone, whereas TIM activity exclusively resides in the fusion protein (Schurig et al., 1995). Cloning the pgk-tim gene from Thermotoga maritima in Escherichia coli, yields monomeric PGK and tetrameric PGK-TIM fusion protein as authentic recombinant proteins (Beaucamp et al., 1995). Both exhibit high intrinsic stability. The thermal transitions at approximately 80 degrees C are irreversible, rendering determination of thermodynamic data impossible. The half-concentrations, (cGdmCl)1/2, of the guanidinium-chloride induced unfolding transitions are 3.0 and 3.9 M GdmCl for PGK and the PGK-TIM fusion protein, respectively. Monitoring denaturation by activity, fluorescence emission and circular dichroism, deactivation and unfolding of the two-domain PGK is found to precede the transitions of the TIM domain. With increasing temperature, (cGdmCl)1/2 is shifted to lower denaturant concentrations; at the same time, the transitions change from bimodal to unimodal. As indicated by the incomplete reversibility of the deactivation/unfolding/dissociation transitions, misfolding, as well as wrong domain interactions seem to interfere with the correct folding and association of the bienzyme complex.

Recombinant hirustasin: production in yeast, crystallization, and interaction with serine proteases.

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

Characterization of the gene encoding Mhp1 from Mycoplasma hyopneumoniae and examination of Mhp1's vaccine potential

The gene encoding Mhp1, a 124 kDa protein from Mycoplasma hyopneumoniae, has been cloned, sequenced, and its product characterized. No significant homology to the gene or encoded polypeptide was found in the Genbank, NBRF, or PIR databases, though this protein appears similar to p97, a putative adhesin of M. hyopneumoniae described by Zhang et al. (Infect. Immun. 63, 1013–1019, 1995) . Two repeated motifs were identified within the 3′ end of the gene and encoded polypeptide. The mhp1 gene was fused to the glutathione S-transferase (GST) gene from Schistosoma japonicum, enabling high-level expression and purification of the protein. Both the authentic and recombinant proteins were recognized by sera from pigs infected with M. hyopneumoniae. In an induced-disease model in pigs, coughing was reduced in animals vaccinated with recombinant GST-Mhp1, although differences were not significant. Only minimal protection against lung lesion formation was provided, and again differences between the Mhp1-vaccinated and nonvaccinated groups were not significant.

The human ENO1 gene product (recombinant human α-enolase) displays characteristics required for a plasminogen binding protein

Plasminogen binds with low affinity in a lysine-dependent manner to many cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an α-enolase-like molecule. The aims of this study were to determine whether recombinant α-enolase (r-α-enolase), encoded by ENO1, was a plasminogen binding protein and to generate polyclonal antibodies against this antigen. Plasminogen specifically bound r-α-enolase with a K d 1.9 μM and approached saturation at 10 μM. Lysine-dependent plasminogen binding to r-α-enolase was demonstrated by a greater than 80% inhibition of binding by the lysine analogues ϵ-amino caproic acid and tranexamic acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-α-enolase with carboxy-peptidase B significantly reduced its plasminogen binding capacity, suggesting that binding required C-terminal lysine residue of r-α-enolase. Binding to r-α-enolase enhanced the activation rate of plasminogen by urokinase but prevented α2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding protein.