Recombinant Human Cytomegalovirus Protease with a C-Terminal (His)6Extension: Purification, Autocatalytic Release of the Mature Enzyme, and Biochemical Characterization

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed inEscherichia coli.The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256–257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)₆tag was expressed at high levels inE. colias inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni²⁺affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256–257 to remove 12 amino acids including the (His)₆tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein inE. coli.In addition, the refolded CMV PR could be crystallized for X-ray diffraction.     

Cloning and Functional Expression of the Mucrosobin Protein, a β-Fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu)

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5′-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His⁴¹, Asp⁸⁴ and Ser¹⁷⁹ in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific β-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquirie a functional snake venom protein with therapeutic potential.     

Expression,purification and characterization of the extracellular domain of human Flt3 ligand in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Fms-like tyrosine kinase 3 ligand (Flt3 ligand, FL) is a cytokine that affects the growth, survival and/or differentiation of hematopoietic cells through the activation of specific tyrosine kinase receptors, and is potentially useful for in vitro HSC amplification. To express the extracellular domain of human Flt3 ligand (hFL^ext) in Escherichia coli, we cloned hFL^ext and constructed the recombinant expression vector pET32a-hFL^ext. hFL^ext was successfully expressed in E. coli as a Trx fusion protein (Trx-hFL^ext) under IPTG (isopropyl-β-d-thiogalactopyranoside) induction for 12 h at 30°C. The Trx-hFL^ext protein, expressed in inclusion bodies even at a low induction temperature, was successfully refolded and purified using dialysis and affinity chromatography. The purified hFL^ext was biologically active and could effectively stimulate the proliferation of mouse bone marrow nucleated cells revealed by cell proliferation assay and colony forming assay. In addition, in synergize with G-CSF and TPO, recombinant purified hFL^ext could stimulate ex vivo expansion of murine Lin⁻Sca-1⁺c-Kit⁺ cells. Therefore, using the E. coli expression system and an affinity chromatography system, we successfully expressed, refolded, and purified a biologically active Trx-hFL^ext protein which might be potentially useful for in vitro HSC amplification.     

Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli

In contrast to mammalian transglutaminases (TGs), plant members of the superfamily are poorly characterized. In order to produce pure and active TG for its functional and structural studies, variants of maize chloroplast transglutaminase (TGZ, Patent WWO03102128) were sub-cloned into a pET28 vector and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were present mainly as insoluble inclusion bodies. The TGZ4p variant with four B-type repeats (M _r∼55 kDa), was affinity purified from urea-solubilized inclusion bodies. TGZ4p was refolded by rapid dilution in a Ca²⁺- and guanidine-containing buffer. Active TGZ4p shows the general catalytic characteristics described for other TGs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression and purification of two major outer membrane proteins from <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a Gram-negative bacterium, is one of Vibrio pathogens common to human and marine animals. Outer membrane proteins of bacteria play an important role during infection and induction of host immune response. In present research, two outer membrane protein genes (OmpK and OmpW) of V. alginolyticus were cloned and expressed. The open reading frames of OmpK and OmpW contain 846 bp and 645 bp, respectively, the mature proteins consist of 261 and 193 amino acid residues. At the signal peptides positions −3 to −1, the amino acids were V-M-A in OmpK and V-F-A in OmpW, which consistented with the observed sequence V-X-A of the signal peptides of transmembrane OMP. The alignment analysis indicated that both proteins were highly conserved, which could serve as surface antigens for vaccine candidates. SDS-PAGE indicated two genes over-expressed in E. coli BL21 (DE3). By affinity chromatography on Ni²⁺-nitriloaceate resin, the recombinant proteins were purified from inclusion bodies. Western blot analysis revealed that both proteins had immunoreactivity, which provided a base for further study on the evaluation of diagnostication and vaccine candidates.     

Purification of the Caenorhabditis elegans Transposase Tc1A Refolded during Gel Filtration Chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT BUFFALO PROLACTIN

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

Overexpression, Purification, and Refolding of a Porphyromonas gingivalis Cysteine Protease from Escherichia coli

This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease.     

Syntheses of Recombinant Yellowtail and Flounder Growth Hormones in Escherichia coli

For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E. coli, expression plasmids were constructed. The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively. These product proteins were accumulated in inclusion bodies in the cells. The recombinant hormones were isolated from the pellets in a glutathione reduction/oxidation buffer. The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity. The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine. The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner.     

Cloning and expression of the polyhydroxyalkanote depolymerase gene from Pseudomonas putida, and characterization of the gene product

A polyhydroxyalkanote (PHA) depolymerase gene ( pha Z) was cloned by PCR from Pseudomonas putida and over-expressed in Escherichia coli as inclusion bodies. Nucleotide sequence analysis predicted an 852 bp open reading frame encoding a protein of 283 amino acids with a predicted molecular weight of 31283 Da. The deduced amino acid sequence had at least 80% homology to the PHA depolymerase from other Pseudomonas strains and consisted a conserved lipase box-like sequence (G-X-S(102)-X-G). The inclusion bodies were refolded and biochemically characterized. The depolymerase activity was optimal at 40 degrees C and pH 8.     

Refolding, purification, and activation of miniplasminogen and microplasminogen isolated from E. coli inclusion bodies

Two des-kringle derivatives of human plasminogen, microplasminogen and miniplasminogen, have been expressed at high levels as inclusion bodies in Escherichia coli using a T7 expression system. In each case, the isolated inclusion bodies were refolded and purified. A final yield of 10% of total refolded protein was observed in each case. Both refolded molecules were successfully activated to their functional forms, microplasmin and miniplasmin, by the plasminogen activator urokinase. The kinetic properties of the refolded microplasmin and miniplasmin were comparable to full length, native plasmin.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for “native” lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Cloning,expression, and characterization of xylose reductase with higher activity from <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene (xyll) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni²⁺-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 μM and 161.9 μM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat=1.44×l0⁴ min⁻¹) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-lA:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L · h xylitol productivity. This XR coupled with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production.     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.     

Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> MA-4680

A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP⁺ as electron acceptor. Apparent K _m values were 4.98 μM for NADP⁺ and 6,620 μM for NAD⁺, respectively, using Mn²⁺ as divalent cation. The enzyme performed a 33,000-fold greater specificity (k _cat/K _m) for NADP⁺ than NAD⁺. Moreover, SaIDH activity was entirely dependent on the presence of Mn²⁺ or Mg²⁺, but was strongly inhibited by Ca²⁺ and Zn²⁺. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP⁺.     

Oxaloacetate decarboxylase of <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>: cloning of genes and expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Archaeoglobus fulgidus harbors three consecutive and one distantly located gene with similarity to the oxaloacetate decarboxylase Na⁺ pump of Klebsiella pneumoniae (KpOadGAB). The water-soluble carboxyltransferase (AfOadA) and the biotin protein (AfOadC) were readily synthesized in Escherichia coli, but the membrane-bound subunits AfOadB and AfOadG were not. AfOadA was affinity purified from inclusion bodies after refolding and AfOadC was affinity purified from the cytosol. Isolated AfOadA catalyzed the carboxyltransfer from [4-¹⁴C]-oxaloacetate to the prosthetic biotin group of AfOadC or the corresponding biotin domain of KpOadA. Conversely, the carboxyltransferase domain of KpOadA exhibited catalytic activity not only with its pertinent biotin domain but also with AfOadC.     

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP⁺. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups. Received: 6 February 1997 / Accepted: 12 June 1997     

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.     

Isolation of the gene and large-scale expression and purification of recombinant Erythrina cristagalli lectin

Using polymerase chain reaction, the coding sequence for Erythrina cristagalli lectin (ECL) has been cloned and expressed in Escherichia coli. The amplified DNA sequence of ECL is highly homologous to that previously reported for Erythrina corallodendron lectin (ECorL), confirming the absence of introns in the ECL gene. The polypeptide sequences of ECL and ECorL have been compared and five amino acids have been identified that differentiate the two proteins. Recombinant E. cristagalli lectin (recECL) was expressed in E. coli from a genomic clone encoding the mature E. cristagalli lectin gene. Constitutive expression localised recombinant protein in inclusion bodies, which were solubilised, and recECL, subsequently refolded and purified by lactose affinity chromatography. Significant advantages were observed for purification from inclusion bodies rather than from a clone optimised to express soluble protein. A large-scale purification scheme has been developed that can prepare functional recECL from inclusion bodies with a yield of 870 mg/l culture. By the range of characterisation methods employed in this study, it has been demonstrated that recECL is functionally equivalent to native ECL obtained from the E. cristagalli plant. In addition, characterisation of the binding of radiolabelled recECL to cultured dorsal root ganglia demonstrated that recECL binds to a single pool of receptors.     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.