Molecular chaperone activity of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) endoplasmic reticulum-located small heat shock protein

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His₆-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recombinant Bacterial Expression of the Lysozyme from the Tobacco-Hornworm <Emphasis Type="Italic">Manduca sexta</Emphasis> with Activity at Low Temperatures

A gene coding for lysozyme from the insect Manduca sexta (Ms-lyz) was expressed in Escherichia coli. The protein was produced as an insoluble cytoplasmic inclusion body which was denatured in 8 M guanidine-HCl, renatured and purified by affinity and ion-exchange chromatography. The N-terminal sequence and the activity of the recombinant protein against Micrococcus luteus confirmed that correct expression had occurred. When Ms-lyz activity was compared to hen egg white lysozyme, the insect lysozyme was active at lower temperatures. These results demonstrate the feasibility of producing a disulfide-bonded lysozyme enzyme in bacteria and suggest that the insect Ms-lyz is an interesting system for further development of an antibacterial functional at low temperatures.     

New DNA polymerase from the hyperthermophilic marine archaeon <Emphasis Type="Italic">Thermococcus thioreducens</Emphasis>

The family B DNA polymerase gene of Thermococcus thioreducens, an archaeon recently isolated from the Rainbow hydrothermal vent field, was cloned and its protein product expressed, purified and characterized. The gene was found to encode a 1,311 amino acid chain including an intein sequence of 537 residues. Phylogenetic analysis revealed a predominantly vertical type of inheritance of the intein in the Thermococcales order. Primary sequence analysis of the mature protein (TthiPolB) showed significant sequence conservation among DNA polymerases in this family. The structural fold of TthiPolB was predicted against the known crystallographic structure of a family B DNA polymerase from Thermococcus gorgonarius, allowing regional domain assignments within the TthiPolB sequence. The recombinant TthiPolB was overexpressed in Escherichia coli and purified for biochemical characterization. Compared with other DNA polymerases from the Thermococcales order, TthiPolB was found to have moderate thermal stability and fidelity, and a high extension rate, consistent with an extremely low K _m corresponding to the dNTP substrate. TthiPolB performed remarkably well in a wide range of PCR conditions, being faster, more stable and more accurate than many commonly used enzymes.     

Purification, characterization, and cloning of trimethylamine dehydrogenase fromMethylophaga sp. strain SK1

Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde, was purified fromMethylophaga sp. strain SK1. The active TMADH was purified 12.3-fold through three purification steps. The optimal pH and temperature for enzyme activity was determined to be 8.5 and 55°C, respectively. TheV _max andK _m values were 7.9 nmol/min/mg protein and 1.5 mM. A genomic DNA of 2,983 bp fromMethylophaga sp. strain SK1 was cloned, and DNA sequencing revealed the open reading frame (ORF) of the gene coding for TMADH. The ORF contained 728 amino acids with extensive identity (82%) to that ofMethylophilus methylotrophus W₃A₁.     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap™ Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris–HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH₄)₂SO_4, 2 mM MgCl_2, 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).     

Cloning and expression of theClostridium thermocellum celS gene inEscherichia coli

Clostridium thermocellum ATCC 27405 produces an extremely complicated multi-component cellulase aggregate (cellulosome) highly active on crystalline cellulose. From the cellulosome, two subunits, CelS (or S _s ;M _r = 82 000) and CelL (or S _l , CipA;M _r = 250 000), have been identified as essential for crystalline cellulose degradation [Wu et al. (1988) Biochemistry 27:1703]. We have determined the DNA sequence of thecelS gene from four cloned DNA fragments encompassing this gene [Wang et al. (1993) J Bacteriol 175:1293]. To express the entirecelS gene inEscherichia coli, thecelS structural gene was amplified by the polymerase chain reaction (PCR) employing the PCR primers corresponding to sequences flanking the desired gene. This PCR product (2.1 x 10³ bases; 2.1 kb) was cloned into anE. coli expression vector pRSET B. Subsequent expression of the cloned gene resulted in a fusion protein (rCelS;M _r = 86 000) as inclusion bodies. The rCelS protein was recognized specifically by an anti-CelS antiserum in a Western blot analysis. The inclusion bodies were purified and solubilized in 5m urea. The refolded rCelS produced very little reducing sugar from carboxymethylcellulose. However, it showed a higher activity on the crystalline cellulose (Avicel) and an even higher activity on phosphoricacid-swollen Avicel. These results indicate that the CelS is an exoglucanase.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation. Project supported by the National Natural Science Foundation of China (Grant No. 39830010).     

Characterization of a dUTPase from the Hyperthermophilic Archaeon <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1 and Its Application in Polymerase Chain Reaction Amplification

Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 (TNA1) revealed the presence of a 471-bp open reading frame with 93% similarity to the dUTPase from Pyrococcus furiosus. The dUTPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dUTP at about a 10-fold higher rate than dCTP. The protein behaved as a dimer in gel filtration chromatography, even though it contains five motifs that are conserved in all homotrimeric dUTPases. The dUTPase showed optimum activity at 80°C and pH 8.0, and it was highly thermostable with a half-life (t _1/2) of 170 min at 95°C. The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl₂, KCl, (NH₄)₂SO₄, and Triton X-100 concentrations, although it was reduced by bovine serum albumin. Addition of the dUTPase to polymerase chain reactions (PCRs) run with TNA1 DNA polymerase significantly increased product yield, overcoming the inhibitory effect of dUTP. Further, addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase. This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases, including Pfu and KOD.     

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His₆-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg⁻¹ protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His₆-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co²⁺ and Mn²⁺ ions. His₆-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k_cat/K_m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s⁻¹ mM⁻¹, respectively.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

A 70-kDa molecular chaperone,DnaK, from the industrial bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: gene cloning,purification and molecular characterization of the recombinant protein

The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purified to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purified BlDnaK was 40°C in the presence of 100 mM KCl. The purified BlDnaK had a V _max of 32.5 nmol Pi/min and a K _M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg²⁺ and K⁺ ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.     

CYP201A2, a cytochrome <Emphasis Type="Italic">P</Emphasis>450 from <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis>, plays a key role in the biodegradation of tributyl phosphate

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in nuclear fuel processing and chemical industries. Rhodopseudomonas palustris, one of the most metabolically versatile photosynthetic bacteria, is shown here to degrade TBP efficiently under photosynthetic conditions. This study shows that this O₂- and NADPH/FMNH₂-dependent process was also catalyzed when TBP was incubated with membrane-associated proteins extracted from this strain. The effects of several regulators of cytochrome P450 activity on the TBP consumption suggest a key role for a cytochrome P450 in this process. Disruption of the rpa0241 gene encoding a putative cytochrome P450 led to a 60% decrease of the TBP catabolism, whereas reintroducing the gene in the mutant restored the wild-type phenotype. The rpa0241 gene was expressed and purified in Escherichia coli. Characterization by UV-visible spectroscopy of the purified recombinant membrane-bound protein (CYP201A2) encoded by the rpa0241 gene revealed typical spectral characteristics of cytochrome P450 with a large spin state change of the heme iron associated with binding of TBP (K _d ≈ 65 μM). It is proposed that CYP201A2 catalyzes the initial step of the biodegradation process of TBP.     

The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Summary The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51669). It was shown by NH₂-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.Abbreviations aa amino acid(s)     

Stimulation of an alpha like DNA polymerase by v-myc related protein of Halobacterium halobium

Partial DNA sequencing of a genomic clone of the archaebacterium Halobacterium halobium, which hydridized with an avian v-myc probe, showed especially the presence, in the organism of one of the conserved regions through myb, myc and adenovirus E1a oncogenes. The archaebacterial deduced amino acid sequence displayed significant homology with the v-myc gene product. In accordance with the partial DNA sequencing which assured a sufficient homology to have similar epitopes, a protein having a molecular weight of 70,000 and possessing high antigenicity with a polyclonal antiserum against avian v-myc protein was isolated and purified from H. halobium extracts. The purified v-myc like protein stimulated in vitro DNA synthesis carried out by the alpha like DNA polymerase of H. halobium.     

Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato

A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5′ to the simian immunodeficiency virus (SIV_mac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by G_M1-ganglioside-enzyme-linked immunosorbent assay (G_M1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by G_M1-ELISA showed that CTB-Gag fusion protein made up approx 0.016–0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.