Effect of proton pump inhibitors on the detection of Helicobacter pylori in gastric biopsies

The adult isoform of human cardiac troponin T (TnT) contains 288 amino acids, 14 of which (4.9%) are encoded by the rarely used arginine codons (12 AGG, 2 AGA) in Escherichia coli genes. To generate sufficient quantity of TnT protein for antibody production, we cloned the corresponding cDNA and expressed it in E. coli. A low-level expression of TnT that comprised only about 1% of total cell protein was initially observed with the use of the native cDNA. The existence of two pairs of consecutive AGG codons AGG(165) AGG(166) and AGG(215) AGG(216) in the cDNA was suspected to be the main cause for this low-level expression. These two pairs of consecutive AGG codons were successively replaced with the major synonymous codon CGT by site-directed mutagenesis. As suspected, a 10-fold increase in TnT expression was obtained when one pair of the rare arginine codons was replaced and a 40-fold increase was achieved when both pairs of the rare codons were replaced. Our finding demonstrates the importance of consecutive rare codons in the suppression of high-level expression of heterologous proteins in E. coli and suggests that in order to maximize protein expression, a similar approach may be taken with other genes which contain consecutive rare codons.     

Production of active recombinant human chymase from a construct containing the enterokinase cleavage site of trypsinogen in place of the native propeptide sequence

Human chymase, a chymotrypsin-like proteinase found in mast cells, was produced in an enzymatically active recombinant form. The protein was expressed in Escherichia coli as part of an insoluble fusion protein which was solubilized and renatured. The structure of the fusion protein was NH2-ubiquitin-enterokinase cleavage site-chymase-COOH. The enterokinase cleavage site of trypsinogen replaced the native propeptide sequence of chymase, allowing for activation by a readily available proteinase (enterokinase) of known specificity. Characterization of refolded-activated recombinant chymase with substrates and inhibitors demonstrated properties identical to that of the native proteinase isolated from skin.     

Purification and activity of a wheat 9-kDa lipid transfer protein expressed in Escherichia coli as a fusion with the maltose binding protein

The cDNA encoding a wheat (Triticum durum) lipid transfer protein of 9 kDa was inserted into an Escherichia coli expression vector, pIH902, and expressed in the bacteria as a fusion with the maltose binding protein. The fusion protein was then purified to homogeneity and subjected to factor Xa cleavage. Although complete cleavage of the fusion protein was obtained, the expected lipid transfer protein was not recovered; it appears to be degraded during protease digestion. However, a fluorescent lipid transfer assay demonstrated that the fusion protein has an activity identical to that of the wheat-purified lipid transfer protein. Thus, this expression system should allow further understanding of the structure/function relationships of wheat lipid transfer proteins.     

The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum

The smallest known intein, found in the ribonucleoside diphosphate reductase gene of Methanobacterium thermoautotrophicum (Mth RIR1 intein), was found to splice poorly in Escherichia coli with the naturally occurring proline residue adjacent to the N-terminal cysteine of the intein. Splicing proficiency increased when this proline was replaced with an alanine residue. However, constructs that displayed efficient N- and C-terminal cleavage were created by replacing either the C-terminal asparagine or N-terminal cysteine of the intein, respectively, with an alanine. Furthermore, these constructs were used to specifically generate complementary reactive groups on protein sequences for use in ligation reactions. Reaction between an intein-generated C-terminal thioester on E. coli maltose-binding protein (43 kDa) and an intein-generated cysteine at the N terminus of either T4 DNA ligase (56 kDa) or thioredoxin (12 kDa) resulted in the ligation of the proteins through a native peptide bond. Thus the smallest of the known inteins is capable of splicing and its unique properties extend the utility of intein-mediated protein ligation to include the in vitro fusion of large, bacterially expressed proteins.     

Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli

A synthetic gene encoding 'core' streptavidin (SAV) [amino acid (aa) residues 13-140 of Streptomyces avidinii SAV] has been efficiently expressed in Escherichia coli from the IPTG-inducible lac promoter of plasmid pET3a. In this system, expression levels are nearly tenfold greater for the synthetic gene than for the corresponding native gene. The synthetic gene was constructed from overlapping oligodeoxyribonucleotides whose sequences were optimized to incorporate codons preferred by highly expressed E. coli genes. Biochemical characterizations by gel methods, aa analysis, N-terminal sequencing, and size exclusion chromatography show that the synthetic gene product purified by affinity chromatography possesses the properties expected for core SAV.     

Basal metabolism of intraperitoneally injected carrier-free 74As-labeled arsenate in rabbits

A streptavidin-RNase H gene fusion was constructed by cloning the Thermus thermophilus RNase H coding sequence in the streptavidin expression vector pTSA18F. The gene was expressed in Escherichia coli, and the resulting fusion protein was purified to apparent homogeneity. The fusion protein was shown to have a molecular weight of 128 kDa and to consist of four subunits. Furthermore, heat treatment of the fusion enzyme showed that it was stable as a tetramer at 65 degrees C. The fusion enzyme was shown to have both biotin binding and RNase H catalytic properties. Using cycling probe technology (CPT), the fusion enzyme was compared to the native RNase H with a biotinylated probe at different ratios of probe:enzyme and varying amounts of synthetic target DNA. At a ratio of 1:1, the fusion enzyme was active in CPT, but the native enzyme was not; both enzymes were active at a 1:5000 ratio of probe:enzyme. The fusion enzyme was further tested using biotinylated and non-biotinylated probes and was shown to be active at a 1:1 ratio with the biotinylated probe but not with the non-biotinylated probe. These experiments show that through binding of the streptavidin-RNase H fusion enzyme to the biotinylated probe, the efficiency of the cycling probe reaction is enhanced.     

Cloning, expression, and properties of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase

cDNA encoding the regulatory subunit of bovine mitochondrial pyruvate dehydrogenase phosphatase (PDPr) has been cloned. Overlapping cDNA fragments were generated by the polymerase chain reaction from bovine genomic DNA and from cDNA synthesized from bovine poly(A)+ RNA and total RNA. The complete cDNA (2885 base pairs) contains an open reading frame of 2634 nucleotides encoding a putative presequence of 31 amino acid residues and a mature protein of 847 residues with a calculated Mr of 95,656. This value is in agreement with the molecular mass of native PDPr (95,800 +/- 200 Da) determined by matrix-assisted laser desorption-ionization mass spectrometry. The mature form of PDPr was expressed in Escherichia coli as a maltose-binding protein fusion, and the recombinant protein was purified to near homogeneity. It exhibited properties characteristic of the native PDPr, including recognition by antibodies against native bovine PDPr, ability to decrease the sensitivity of the catalytic subunit to Mg2+, and reversal of this inhibitory effect by the polyamine spermine. A BLAST search of protein data bases revealed that PDPr is distantly related to the mitochondrial flavoprotein dimethylglycine dehydrogenase, which functions in choline degradation.     

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Expression and Characterization of the Intracellular Part of Receptor Protein Tyrosine Phosphatase PTPRU

PTPRU is an MAM domain-containing receptor-like protein tyrosine phosphatase. Previous studies have demonstrated an important role of the enzyme in the maintenance of epithelial integrity and in the regulation of the Wnt/β-catenin signaling pathway. To better understand the function of PTPRU, we cloned and expressed the intra- cellular portion of PTPRU as a GST fusion protein in E. coli cells. We purified the protein to homogeneity and used it to immunize mice for antibody production. The resultant antibody specifically recognized PTPRU over-expressed in the cell line. Western blot analyses demonstrated the partition of truncated forms of PTPRU containing the cadhe- rin-like domain in the Triton X-100-insoluble fraction, and immunofluorescent cell staining revealed the localization of these proteins in punctate intracellular structures. Our data suggest that the cadhefin-like domain of PTPRU has a major role in determining its intracellular localization.     

Clinical gait analysis in a rehabilitation context: some controversial issues

The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin alpha-sarcin to be used for immunoconjugate production. alpha-Sarcin cDNA was isolated from Aspergillus giganteus strain MDH 18,894 and its expression in Escherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant alpha-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernatant. A variant form of alpha-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.     

Gene cloning, DNA sequencing, and expression of thermostable beta-mannanase from Bacillus stearothermophilus

A DNA genomic library constructed from Bacillus stearothermophilus, a gram-positive, facultative thermophilic aerobe that secretes a thermostable beta-mannanase, was screened for mannan hydrolytic activity. Recombinant beta-mannanase activity was detected on the basis of the clearing of halos around Escherichia coli colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum. The nucleotide sequence of the mannanase gene, manF, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da. From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases. The manF coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in E. coli as a recombinant fusion protein containing a hexahistidine N-terminal sequence. The fusion protein has thermostability similar to the native enzyme and was purified by Ni2+ affinity chromatography. The values for the kinetic parameters Vmax and Km were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme.     

Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands

A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.     

Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments

In this report expression of the biologically active N-terminal half (amino acids 1-153) of thrombopoietin (TPO153) in Escherichia coli is described and the structure-function relationships in TPO are explored. TPO153 was chosen for expression because of its full biological activity. Since natural TPO153 cDNA expressed poorly, synthetic cDNA was constructed with a unique polymerase chain reaction to enhance the expression. In addition, the 5'-end codons of the synthetic cDNA were altered to maximize the expression. The expressed TPO153 was refolded and then purified to homogeneity. The protein is biologically active, and interestingly, the EC50 of this protein is 8-10-fold smaller in a TPO-dependent cell proliferation assay than that of full-length wild-type TPO. In order to identify the amino acid residues that are involved in the interaction between TPO and its receptor, all charged residues and some of the uncharged residues on the four putative helices of TPO were mutated and biological activities of the mutant proteins were examined. The mutagenesis studies suggest that there are at least two clusters of residues that are vital for TPO to be able to interact with its receptor. These residues are centred respectively around arginine 10 on helix 1 and around lysine 138 on helix IV. The successful expression of the protein in E. coli will greatly facilitate biochemical and crystallographic studies of TPO, and the structure-function relationship studies suggest that TPO has two binding sites which may interact with two individual receptors, resulting in dimerization of the receptors.     

Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion

The GnRH receptor (GnRHR) is a G protein-coupled receptor expressed by gonadotropes in the anterior pituitary gland. In the past several years, much has been learned about the structure-function relationships that exist in this receptor with regard to ligand binding and signal transduction. However, the lack of specific antibodies has precluded any analyses of the behavior of the unbound form of this receptor. We have constructed a functional GnRHR in which enhanced green fluorescent protein is fused to the carboxyl-terminus of the murine GnRHR. This fusion receptor was expressed diffusely throughout the cell, with approximately 38% of the fusion receptors colocalized with a plasma membrane marker in the gonadotrope-derived alphaT3 cell line, and approximately 82% of the fusion receptors colocalized with a membrane marker in Chinese hamster ovary cells. Furthermore, the fusion receptor displayed a Kd of 0.8 nM for iodinated des-Gly10,D-Ala-6-GnRH N-ethyl amide in Chinese hamster ovary cells, which was similar to the Kd of the native GnRHR expressed in alphaT3 cells. The surface mobility of the fusion protein was examined by fluorescence photobleaching recovery methods. In the unbound state the majority of the receptors were laterally mobile and displayed a lateral diffusion rate of 1.2-1.6 x 10(-9) cm2/sec. Binding of GnRH reduced the rate of lateral diffusion over 3-fold and reduced the fraction of mobile receptors from approximately 76-91% to 44-61%. Like GnRH, the competitive GnRH antagonist antide slowed the rate of receptor diffusion approximately 3-fold. In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Thus, an intrinsically fluorescent GnRHR is trafficked to the plasma membrane of mammalian cells, is capable of ligand binding and signal transduction, and allows direct observation of the GnRHR in the nonligand-bound state. Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist.     

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.     

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.     

Relation of arterial geometry to luminal narrowing and histologic markers for plaque vulnerability: the remodeling paradox

The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC, to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr+ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr+ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus, and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.     

Expression of active human tissue-type plasminogen activator in Escherichia coli

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 microgram of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding.