Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.

The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.     

Purification and characterization of recombinant single-chain urokinase produced in Escherichia coli

Recombinant single-chain urokinase (rUK1) has been purified from Escherichia coli. The purification utilizes a refractile body purification, followed by batch DE-52 cellulose extraction, hydroxylapatite chromatography, and S-200 chromatography. Two-chain rUK (rUK2) is separated from rUK1 on benzamidine--Sepharose. The purification eliminates proteases early in the procedure so the rUK1 will not be cleaved to rUK2. The rUK1 has been characterized by amino-terminal analysis as well as carboxy-terminal analysis after cleavage by plasmin.     

Purification and characterization of recombinant bovine growth hormone produced in Escherichia coli

Using the pUBJ10 plasmid containing the modified bovine growth hormone (bGH) cDNA, large production has been attempted in E. coli BL21 strain. The bGH was highly expressed upto the level of 35% of total cell proteins by IPTG induction and temperature shift to 40°C. The recombinant bGH (rbGH) was isolated from inclusion bodies by solubilization in 10 M urea and followed by DEAE-TOYOPEARL 650C ion exchange and Sephadex G-100 column chromatography. The pUBJ10-derived bGH was eluted at 25.28 min similar to the standard bGH (at 25.18 min) by reverse-phase HPLC. The analysis of N-terminal amino acid showed that the mature bGH has glutamic acid as a first amino acid in agreement with DNA sequencing data. The biological activity was indirectly measured by radioreceptor assay and compared with a pituitary-derived bGH.     

Purification and characterization of recombinant human interleukin-2 produced in Escherichia coli

Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli was purified to apparent homogeneity by cation exchange chromatography and reverse phase high performance liquid chromatography. The amino acid composition, amino terminal amino acid sequence, and carboxyl terminal amino acid were consistent with those deduced from the cDNA sequence. Besides the molecular species with the amino terminal Ala, the purified preparation contained another species having an additional Met residue at the amino terminus corresponding to the initiation codon AUG. The molar absorption coefficient of rIL-2 was determined to be 9.58 X 10(3) M-1 cm-1 at 280nm in water. Ultracentrifugal analyses revealed that it existed as a monomeric form in 0.1 M NaCl. The apparent sedimentation coefficient (S20,w) was calculated to be 1.8 S.     

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.     

Purification and characterization of recombinant human interleukin-1 beta produced in Escherichia coli

Recombinant human interleukin-1 beta (rIL-1 beta) produced in Escherichia coli was purified to homogeneity by a combination of mass ion exchange column chromatography, ion exchange and gel filtration high performance liquid chromatography. The purified rIL-1 beta had a molecular weight of 18 kD on SDS-polyacrylamide gel electrophoresis and an isoelectric point of 6.9 on analytical isoelectric focusing. These values were almost same as those of natural interleukin-1 beta. The amino acid composition and amino acid sequence of the amino terminal region were consistent with those deduced from the cDNA sequence. In addition, the primary structure was confirmed by peptide mapping with lysyl-endopeptidase on reverse phase HPLC. Besides rIL-1 beta with amino terminal Ala, two molecular species, [Met0] rIL-1 beta and [desAla1] rIL-1 beta, were also obtained. Biological and physicochemical properties of the three species of rIL-1 beta were compared.     

Purification and characterization of progenipoietins produced in Escherichia. coli

The progenipoietins (ProGPs) are a family of genetically engineered chimeric proteins that contain receptor agonist activity for both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. These unique proteins have previously been shown to induce the proliferation of multiple cell lineages. The characterization of two progenipoietins, ProGP-1 and ProGP-4, refolded and purified from an Escherichia coli expression system is described. These ProGP molecules differ in the orientation of the two receptor agonists and, in addition, ProGP-4 contains a fetal liver tyrosine kinase-3 receptor agonist that has been circularly permuted to modulate its activity. Static light scattering analyses demonstrated that both ProGP molecules exist as dimers, most likely through non-covalent interaction of the fetal liver tyrosine kinase-3 receptor agonist domains. ProGP-1 and ProGP-4 have comparable secondary structures, as analyzed by circular dichroism; however, their tertiary structures, as measured by intrinsic fluorescence, were demonstrated to be different. Differential scanning calorimetry demonstrated that the thermal stability of these two proteins was indistinguishable. Interestingly, these dual agonist proteins yielded only a single melting temperature value that was intermediate between that of their individual receptor agonist components, indicating that these chimeric molecules behave as a single domain protein during thermal denaturation. This study describes the purification and physico-chemical properties of this class of proteins generated using an E. coli expression system.     

重组大肠杆菌右旋糖酐蔗糖酶的纯化及其性质

[目的]研究工程菌E.coli BL21(DE3)/pET28-dexYG产右旋糖酐蔗糖酶的纯化和酶学性质.[方法]工程菌经过IPTG诱导后生产含His-tag融合蛋白的右旋糖酐蔗糖酶,通过硫酸铵沉淀、Ni-NTA亲和层析纯化,得到纯度较高的酶蛋白,并对纯酶进行了酶学性质及动力学研究.[结果]经过SDS-PAGE测得该酶的分子量约为170 kDa,与理论推测值基本相同.以蔗糖为底物,酶促反应的最适温度为25～30℃,最适pH值为5.4,动力学常数Km值为10.43 mmol/L;酶活在pH 5.0～8.0较为稳定,在室温(25 ℃)保藏4天仍有59%的酶活力,4℃保存7周酶活力仅下降一半,但在35℃以上失活很快;Ca2 对催化作用有较大的促进,Mg2 有微弱的促进作用,K 对催化反应无影响,Cu2 的抑制作用最强.其他试剂对重组酶的活性有不同程度的影响,其中SDS抑制作用很强.[结论]研究为重组右旋糖酐蔗糖酶纯酶的获取、得到稳定性好、活性高的酶反应体系及利用该酶进行催化反应和工业化应用提供了重要参数.     

Nucleotide sequence and molecular characterization of a dextranase gene from Streptococcus downei

DNA fragments encoding the Streptococcus downei dextranase were amplified by PCR and inverse PCR based on a comparison of the dextranase gene (dex) sequences from S. sobrinus, S. mutans, and S. salivarius, and the complete nucleotide sequence of the S. downei dex was determined. An open reading frame (ORF) of dex was 3,891 bp long. It encoded a dextranase protein (Dex) consisting of 1,297 amino acids with a molecular mass of 139,743 Da and an isoelectric point of 4.49. The deduced amino acid sequence of S. downei Dex had homology to those of S. sobrinus, S. mutans and S. salivanus Dex in the conserved region (made of about 540 amino acid residues). DNA hybridization analysis showed that a dex DNA probe of S. downei hybridized to the chromosomal DNA of S. sobrinus as well as that of S. downei, but did not to other species of mutans streptococci. The C terminus of the S. downei Dex had a membrane-anchor region which has been reported as a common structure of C termini of both the S. mutans and S. sobrinus Dex. The recombinant plasmid which harbored the dex ORF of S. downei produced a recombinant Dex enzyme in Escherichia coli cells. The analysis of the recombinant enzyme on SDS-PAGE containing blue dextran showed multiple active forms as well as dextranases of S. mutans, S. sobrinus and S. salivarius.     

Overproduction of a dextranase inhibitor by Streptococcus sobrinus mutants.

An inhibitor of Streptococcus sobrinus endodextranase was detected in the extracellular fractions of UAB66 mutants identified following ethyl methanesulfonate mutagenesis as either devoid of dextranase activity (Dex-) or overproducing water-soluble glucan. The two groups of mutants had the same phenotype and displayed no dextranase activity in assays of extracellular fractions (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 34:1044-1055, 1981) and had been shown to be defective in adherence (Adh-) and capable of inhibiting adherence of wild-type strains during cocultivation in vitro (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 50:826-832, 1985) and in vivo in gnotobiotic rats (K. Takada, T. Shiota, R. Curtiss III, and S. M. Michalek, Infect. Immun. 50:833-843, 1985). By analysis of proteins in Western blots (immunoblots) and following blue dextran-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDS-PAGE), it was demonstrated that these Dex- mutants did synthesize enzymatically active dextranase. From the results of mixing experiments, it was determined that these Dex- Adh- mutants produced enhanced amounts of a cell surface-localized or a cell-associated dextranase inhibitor (Dei). Dei was heat stable but trypsin sensitive. By adding excess dextranase following BD-SDS-PAGE, Dei was detected as blue bands with apparent molecular masses of 43, 40, 37, 27, and 23 kDa. Dei competitively inhibits dextranase activity and is synthesized by wild-type S. sobrinus strains, with the amount varying depending upon growth medium and stage in the growth cycle. R. M. Hamelik and M. M. McCabe (Biochem. Biophys. Res. Commun. 106:875-880, 1982) previously described a Dei in a wild-type S. sobrinus strain.     

Cloning and DNA sequencing of the dextranase inhibitor gene (dei) from Streptococcus sobrinus.

Some dextranase-deficient (Dex-) mutants of Streptococcus sobrinus UAB66 (serotype g) synthesize a substance which inhibits dextranase activity (S.-Y. Wanda, A. Camilli, H. M. Murchison, and R. Curtiss III, J. Bacteriol. 176:7206-7212, 1994). This substance produced by the Dex- mutant UAB108 was designated dextranase inhibitor (Dei) and identified as a protein. The Dei gene (dei) from UAB108 has been cloned into pACYC184 to yield pYA2651, which was then used to generate several subclones (pYA2653 to pYA2657). The DNA sequence of dei was determined by using Tn5seq1 transposon mutagenesis of pYA2653. The open reading frame of dei is 990 bp long. It encodes a signal peptide of 38 amino acids and a mature Dei protein of 292 amino acids with a molecular weight of 31,372. The deduced amino acid sequence of Dei shows various degrees of similarity with glucosyltransferases and glucan-binding protein and contains A and C repeating units probably involved in glucan binding. Southern hybridization results showed that the dei probe from UAB108 hybridized to the same-size fragment in S. sobrinus (serotype d and g) DNA, to a different-size fragment in S. downei (serotype h) and S. cricetus (serotype a), and not at all to DNAs from other mutans group of streptococci.     

Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.     

Purification and characterization of recombinant cell surface protein antigen A of Streptococcus sobrinus B13N.

1. It has been reported that immunization of rhesus monkeys with the surface protein antigen I/II from Streptococcus mutans significantly reduced dental caries. 2. The surface protein antigen A (SpaA) from Streptococcus sobrinus is known to correspond antigenically to I/II. MD51 is an Escherichia coli host containing pMD51, a plasmid encoding the SpaA gene from Streptococcus sobrinus B13N. 3. The recombinant SpaA (rSpaA) was purified from cell extracts of Escherichia coli clone MD51. 4. The purified recombinant SpaA was homogeneous with a molecular weight of 210 kDa according to SDS-PAGE and had an isoelectric point of 4.2 based on isoelectric focusing. 5. Amino acid composition of rSpaA showed a relatively high amount of hydrophobic amino acids (39.7%).     

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli.

The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE-Sepharose CL-6B, S-Sepharose, and MonoQ-Sepharose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase was active on levan, inulin, and sucrose with Km values of 1.2 microM, 6.8 mM, and 65 mM, respectively. The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55 degrees C. Levanase was rapidly inactivated at 60 degrees C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme activity was completely inactivated by Ag+ and Hg2+ ions, indicating that a sulfhydryl group is involved. A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography-low-angle laser light scattering-interferometric differential refractive index techniques. This indicates that levanase is an exoenzyme acting by the single-chain mode.     

Purification and properties of recombinant rat catalase produced in Escherichia coli

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.     

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.     

Purification and characterization of recombinant sTRAIL expressed in Escherichia coli

As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) has drawn considerable attention. This report presented the purification and characterization ofsoluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized andrefolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purifiedto electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purifiedsTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with EDs0 about 1.5mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had astructure similar to that of native protein with 13-sheet secondary structure. This efficient procedure ofsTRAIL renaturation may be useful for the mass production of this therapeutically important protein.     

Preparation and characterization of bovine growth hormones produced in recombinant Escherichia coli.

Two analogues of bovine growth hormone (BGH) have been produced in Escherichia coli by recombinant DNA techniques. In analogue Delta-1, the N-terminal alanine residue of the full-length bovine sequence is replaced by methionine. In analogue Delta-9, which is expressed at much higher levels than is Delta-1, the full-length bovine sequence is truncated at the N-terminus by eight residues and there is a serine-for-glycine substitution in the first position of the truncated protein. Both analogues, which were characterized by isoelectric focusing (i.e.f.), polyacrylamide-gel electrophoresis in the presence of SDS (SDS/PAGE), amino acid analysis and N-terminal amino acid sequence determination using combined g.l.c.-m.s., are compared with BGH isolated from pituitaries. In contrast with pituitary-derived BGH, the recombinant-derived proteins are homogeneous on SDS/PAGE and on i.e.f. In a radioimmunoassay, a radioreceptor assay and a bioassay in vivo (rat tibia), Delta-9 BGH showed very similar characteristics to the pituitary-derived hormone. Similar results have also been obtained with the Delta-1 analogue.     

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.