Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.9 (enterokinase)
675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5, by heterologous expression in Escherichia coli is described. The genes encoding mature bacteriocin were cloned into an E. coli expression system and expressed as a fusion protein with a thermostable thioredoxin. Recombinant E. coli were cultivated following a fed-batch fermentation process with pH, temperature and oxygenation regulation. The overexpression of the fusion proteins was improved by replacing IPTG by lactose. The fusion proteins were purified by thermal coagulation followed by affinity chromatography. The thioredoxin fusion protein was removed by using CNBr instead of enterokinase and the carnobacteriocins were recovered by reverse-phase chromatography. These optimizations led us to produce up to 320 mg of pure protein per liter of culture, which is four to ten fold higher than what is described for other heterologous expression systems.
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PMID:Optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 by heterologous expression in Escherichia coli. 1831 33

This work reports the first successful recombinant expression and purification of human beta-defensin 5 (HBD5) and human beta-defensin 6 (HBD6) in Escherichia coli. HBD5 and HBD6 are cationic antimicrobial peptides with three conserved cysteine disulfide bonds. Two codon-optimized sequences coding the HBD5 gene (sHBD5) and HBD6 gene (sHBD6), respectively, were synthesized, and each gene fused with thioredoxin A (TrxA) to construct the expression vectors. The plasmids were transformed into E. coli BL21 (DE3) strains and cultured in MBL medium, which gave high volumetric productivity of HBD5 and HBD6 fusion proteins of up to 1.49 g L(-1) and 1.57 g L(-1), respectively. Soluble HBD5 and HBD6 fusion proteins account for 95.2% and 97.6% of the total fusion proteins, respectively. After cell disruption, the soluble fusion proteins were recovered by affinity chromatography and cleaved by enterokinase. Pure HBD5 and HBD6 were recovered using cationic exchange chromatography. The overall recoveries of HBD5 and HBD6 were 38% and 35%, respectively. Importantly, both HBD5 and HBD6 products showed antimicrobial activity against E. coli but not Staphylococcus aureus. Antimicrobial activity against E. coli of both HBD5 and HBD6 were suppressed by NaCl.
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PMID:Production of bioactive human beta-defensin 5 and 6 in Escherichia coli by soluble fusion expression. 1859 35

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.
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PMID:Expression and purification of soluble B lymphocyte stimulator from recombinant Escherichia coli. 1866 61

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1-155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.
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PMID:Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2). 1926 79

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. In this paper, we report the expression, purification, and preparation of a recombinant form of the extracelluar domain of the TRAIL (sTRAIL) without posttranslational modifications, which may selectively induce apoptosis of tumor cells in vitro. To obtain recombinant nonfusion sTRAIL protein, the encoding region for sTRAIL was cloned between KpnI and BamHI in pET32a. The Trx (thioredoxin)/sTRAIL fusion proteins were expressed in the form of inclusion bodies in Escherichia coli host strain BL21 (DE3). The expression level was more than 35% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2 M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >98% were obtained. Trx/sTRAIL L proteins were digested by enterokinase to both Trx and sTRAIL fragments, which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rsTRAIL (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the recombinant sTRAIL may have cancer therapeutic applications.
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PMID:High-level expression, purification, and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). 1930 78

This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l(-1), respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae.
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PMID:Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27. 1937 62

Histo-aspartic protease (HAP) from Plasmodium falciparum is an intriguing aspartic protease due to its unique structure. Our previous study reported the first recombinant expression of soluble HAP, in its truncated form (lys77p-Leu328) (p denotes prosegment), as a thioredoxin (Trx) fusion protein Trx-tHAP. The present study found that the recombinant Trx-tHAP fusion protein aggregated during purification which could be prevented through the addition of 0.2% CHAPS. Trx-tHAP fusion protein was processed into a mature form of tHAP (mtHAP) by both autoactivation, and activation with either enterokinase or plasmepsin II. Using gel filtration chromatography as well as sedimentation velocity and equilibrium ultracentrifugation, it was shown that the recombinant mtHAP exists in a dynamic monomer-dimer equilibrium with an increasing dissociation constant in the presence of CHAPS. Enzymatic activity data indicated that HAP was most active as a monomer. The dominant monomeric form showed a K(m) of 2.0 microM and a turnover number, k(cat), of 0.036s(-1) using the internally quenched fluorescent synthetic peptide substrate EDANS-CO-CH(2)-CH(2)-CO-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-Dap-(DABCYL)-OH (2837b) at pH 5.2.
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PMID:Characterization of the monomer-dimer equilibrium of recombinant histo-aspartic protease from Plasmodium falciparum. 2043 72

TGFbeta signaling is initiated by binding of growth factor ligand to two related single-pass transmembrane receptor serine/threonine kinases, known as the TGFbeta type I (TbetaRI) and type II (TbetaRII-ED) receptors. TbetaRII-ED is essential for all TGFbeta-induced signals. The DNA sequence encoding the extracellular domain of human TbetaRII-ED (TbetaRII-ED, residues 4-136) was synthesized from 20 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. High level expression ( approximately 1 gL(-1)) of thioredoxin/TbetaRII-ED fusion was achieved in Escherichia coli BL21(DE3) strain mainly in soluble form. The soluble thioredoxin/TbetaRII-ED fusion has been purified and refolded on Ni-NTA agarose. After cleavage of purified thioredoxin/TbetaRII-ED fusion by recombinant human enteropeptidase light chain (L-HEP) the target protein of TbetaRII-ED was separated from thioredoxin on Ni-NTA agarose. Fourteen milligrams of highly purified TbetaRII-ED without N- or C-terminal tags was yielded from 100mL cell culture. The purified preparation of TbetaRII-ED was highly homogenous, as shown by SDS-PAGE with silver staining, HPLC and mass spectroscopy analysis. The binding of TbetaRII-ED purified from E. coli to TGFbeta1 was shown to be comparable to commercial material purified from NSO cells. Recombinant TbetaRII-ED could be employed as an antagonist of TGFbeta1 and TGFbeta3 in vitro and in vivo as well as for therapy of fibrotic disorders and some types of cancer.
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PMID:An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFbeta type II receptor. 2045 68

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the beta (1-4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc-hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc-hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc-hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc-hly may have important potential application as a future safely administered human drug and food additive.
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PMID:Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli. 2049 32

Human alpha-defensin 6 (HD(6)), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD(6) through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD(6) was primarily constructed by inserting a PCR fragment encoding mature HD(6) peptide (mHD(6)) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD(6) fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD(6) (rmHD(6)) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD(6), followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD(6) with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD(6) possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD(6).
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PMID:High efficiency preparation of bioactive human alpha-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression. 2054 2


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