Expression, purification, and characterization of recombinant fibulin-5 in a prokaryote expression system

Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N²⁺-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.     

Expression and characterization of recombinant bovine lactoferrin in E. coli

Lactoferrin is a member of the transferrin family of iron-binding proteins with a number of properties, including antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria. bovine lactoferrin cDNA was isolated, cloned and expressed as a fusion protein. The amino acid sequence of the fusion was analyzed and compared with other species. Crystallographic data were used to compare structural differences between bovine and human lactoferrin in 3-D models. A thioredoxin fusion protein was expressed and shown to have a different molecular weight compared with native bLf. After purification using Ni-NTA, the yield of recombinant bovine lactoferrin was 15.3 mg/l with a purity of 90.3 %. Recombinant bLf and pepsin-digested rbLf peptides demonstrated antibacterial activity of 79.8 and 86.9 %, respectively. The successful expression of functional, active and intact rbLf allows us to study the biochemical interactions of antimicrobial proteins and peptides and will facilitate their study as immunomodulators.     

Expression and characterization of bovine lactoperoxidase by recombinant baculovirus

Lactoperoxidase (LPO) is a heme-containing oxidation-reduction enzyme present in milk. In this study, the gene encoding bovine lactoperoxidase (bLPO) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLPO was isolated. A bLPO-related recombinant baculovirus-expressed protein of 78 kDa was detected using anti-bLPO antibodies. After digestion with N-glycosidase F, the molecular weight of the recombinant bLPO (rbLPO) decreased. In addition, rbLPO reacted with lectin, indicating that the protein was glycosylated. The rbLPO activity and heme content in the culture supernatants increased upon addition of delta-aminolevulinic acid, which is a heme precursor. Differences in the delta-aminolevulinic acid-dependent circular dichroism spectrum and rbLPO pepsin hydrolysis were observed. These results suggest that the secondary structure and structural stability of rbLPO depends on the heme environment. Our data suggest that this bLPO expression system is useful for studying structure, catalytic mechanisms, and biological function.     

Expression and characterization of bovine lactoperoxidase by recombinant vaccinia virus

Lactoperoxidase (LPO) is a 78 kDa heme-containing oxidation–reduction enzyme present in milk, found in physiological fluids of mammals. LPO has an antimicrobial activity, and presumably contribute to the protective functions of milk against infectious diseases. In this study, recombinant vaccinia virus expressing bovine LPO (vv/bLPO) was constructed. In rabbit kidney (RK13) cells infected with vv/bLPO, recombinant bLPO was detected in both cell extracts and culture supernatants. Tunicamycin treatment decreased the molecular weight of recombinant bLPO, indicating that recombinant bLPO contains a N-linked glycosylation site. The replication of recombinant vaccinia viruses expressing bovine lactoferrin (vv/bLF) at a multiplicity of infection (moi) of 5 plaque-forming units (PFU)/cell was inhibited by antiviral activity of recombinant bLF, suggesting that vv/bLF has an antiviral effect against vaccinia virus. On the other hand, the replication of vv/bLPO at a moi of 5 PFU/cell was not inhibited by antiviral activity of recombinant bLPO, indicating that this recombinant virus could be used as a suitable viral vector. These results indicate that a combination of bLPO and vaccinia virus vector may be useful for medical and veterinary applications in vivo.     

Expression and structural characterization of the recombinant human doppel protein

The doppel protein (Dpl) is a newly recognized prion protein (PrP)-like molecule encoded by a novel gene locus, prnd, located on the same chromosome as the PrP gene. To study the structural features of Dpl, we have expressed recombinant human Dpl corresponding to the putative mature protein domain (residues 24-152) in Escherichia coli. The primary structure of the recombinant Dpl 24-152 was characterized using gel electrophoresis, N-terminal Edman sequencing, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization mass spectrometry. Dpl 24-152 was shown to contain two disulfide bonds (Cys94-Cys145 and Cys108-Cys140). The secondary structure of Dpl was analyzed using far-UV circular dichroism spectroscopy. Dpl 24-152 was found to be an alpha-helical protein having a high helical content (40%). Dpl 24-152 exhibited characteristics of a thermodynamically stable protein that undergoes reversible and cooperative thermal denaturation. In addition, Dpl was found to be soluble and sensitive to proteinase K digestion. Therefore, Dpl 24-152 possesses biochemical properties similar to those of recombinant PrP. This study provides knowledge about the molecular features of human Dpl that will be useful in further investigation into its normal function and the role it may play in neurodegenerative diseases.     

Arachidonic acid epoxygenase: structural characterization and quantification of epoxyeicosatrienoates in plasma.

Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase.     

Expression of recombinant chinese bovine enterokinase catalytic subunit in P. pastoris and its purification and characterization

EK (enterokinase) is a serine proteinase which consistsof a heavy chain and a light chain linked by a disulfidebond. The light chain of EK contains a chymotrypsin-likeserine proteinase domain sufficient for the normal recog-nition and cleavage of EK subst…     

Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin

The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.     

褐藻胶裂解酶基因的克隆表达及重组酶酶学性质

【目的】克隆交替假单胞菌(Pseudoalteromonas sp.)BYS-2的褐藻胶裂解酶基因,实现其在大肠杆菌细胞中异源表达,对分离纯化的重组酶进行酶学性质研究。【方法】以交替假单胞菌BYS-2菌株基因组DNA为模板,克隆得到褐藻胶裂解酶基因alg738,构建重组基因工程菌BL21(DE3)/p ET22b-alg738,诱导表达,表达产物通过Ni-NTA树脂纯化后进行酶学性质研究。【结果】重组酶的最适反应p H为8.0,在p H 6.0-9.0范围内37°C保温1 h仍能保持84%以上的相对酶活力,具有较好的p H稳定性;最适反应温度为45°C,热稳定性实验显示在37°C下保温60 min其残余酶活力仍达66.6%;在5 mmol/L浓度下,Na~+、Mg~(2+)、Mn~(2+)对该酶具有明显的促进作用,Ni~(2+)、Co~(2+)、Cu~(2+)、Hg~(2+)、Zn~(2+)、EDTA、β-巯基乙醇、SDS具有明显的抑制作用。动力学参数Km、Vmax分别为1.11 g/L和0.011 g/(L·min),底物特异性分析表明该重组酶为偏好聚甘露糖醛酸钠(Poly M)裂解作用的双功能酶。【结论】重组褐藻胶裂解酶具有良好的酶学特性,为褐藻胶裂解酶的开发应用打下基础。     

Inhibition of prokaryote and eukaryote cells adhesion by sea proteobacteria Pseudoalteromonas nigrifaciens KMM 156 lipopolysaccharide and its fragments]

The impact of Pseudoalteromonas nigrifaciens KMM 156 lipopolysaccharide and its fragments on adhesion of prokaryote and eukaryote cells was studied. The lipopolysaccharide and O-specific polysaccharide identical by its structure to capsular polysaccharide lowered the number of the Yersinia pseudotuberculosis bacteria attached to sheep erythrocytes and adhesion of mouse neutrophils to plastic. The kora oligosaccharide had no effect on the processes.     

Expression and characterization of recombinant murine cytomegalovirus protease.

The protease domain of the murine cytomegalovirus (MCMV) M80 open reading frame was expressed in and purified from Escherichia coli. The recombinant enzyme was recovered as a mixture of active one- and two-chain forms. The two-chain enzyme was formed by internal cleavage of the one-chain enzyme at the I site. Activity measurements showed that MCMV protease cleaves R- and M-site peptide mimics with kinetics similar to those of recombinant human cytomegalovirus (HCMV) protease. Both the MCMV and HCMV proteases cleave I-site peptide substrates very poorly, but the crystal structure of the HCMV protease indicates that the cytomegalovirus I site likely resides on a solvent-exposed loop close to the active site.     

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.     

Genetic variants of bovine beta-lactoglobulin. A novel wild-type beta-lactoglobulin W and its primary sequence

A novel bovine beta-lactoglobulin W has been isolated and its complete primary structure is presented. It was isolated by chromatofocusing of a beta-lactoglobulin AW heterozygote and purified by recrystallization. During sequencing of the oxidized protein, it became evident that the new beta-lactoglobulin W is a subtype of variant B with a single difference at position 56. This Ile----Leu substitution, which means a shift of a methyl group from C-beta to C-gamma of the amino-acid side chain causes a change of pI of 0.007 units, which can be detected by high resolution electrophoresis. This Ile56 amino-acid residue is among the most conserved residues with the exception of kangaroo beta-LG. The structures of other bovine beta-lactoglobulins and their relationships are discussed.     

Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase

We describe the heterologous expression of a recombinant Saccharomyces cerevisiae isoleucyl-tRNA synthetase (IRS) gene in Escherichia coli, as well as the purification and characterization of the recombinant gene product. High level expression of the yeast isoleucyl-tRNA synthetase gene was facilitated by site-specific mutagenesis. The putative ribosome-binding site of the yeast IRS gene was made to be the consensus of many highly expressed genes of E. coli. Mutagenesis simultaneously created a unique BclI restriction site such that the gene coding region could be conveniently subcloned as a "cassette." The variant gene was cloned into the expression vector pKK223-3 (Brosius, J., and Holy, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6929-6933) thereby creating the plasmid pKR4 in which yeast IRS expression is under the control of the isopropyl-thio-beta-galactopyranoside (IPTG)-inducible tac promoter. Recombinant yeast IRS, on the order of 10 mg/liter of cell culture, was purified from pKR4-infected and IPTG-induced E. coli strain TG2. Yeast IRS was purified to homogeneity by a combination of anion-exchange and hydroxyapatite gel chromatography. Inhibition of yeast IRS activity by the antibiotic pseudomonic acid A was tested. The yeast IRS enzyme was found to be 10(4) times less sensitive to inhibition by pseudomonic acid A (Ki = 1.5 x 10(-5) M) than the E. coli enzyme. E. coli strain TG2 infected with pKR4, and induced with IPTG, had a plating efficiency of 100% at inhibitor concentrations in excess of 25 micrograms/ml. At the same concentration of pseudomonic acid A, E. coli strain TG2 infected with pKK223-3 had a plating efficiency less than 1%. The ability of yeast IRS to rescue E. coli from pseudomonic acid A suggests that the eukaryotic synthetase has full activity in its prokaryotic host and has specificity for E. coli tRNA(ile).     

Expression and characterization of recombinant gamma-tryptase

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. gamma-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. gamma-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys(26) and Cys(145), that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed gamma-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd gamma-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc gamma-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of gamma-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that gamma-tryptase possesses a substrate preference and inhibitor profile distinct from that of beta-tryptase. Although the role of gamma-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of beta-tryptase.