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)

Enterokinase (EC 3.4.21.9) is a serine proteinase in the duodenum that exhibits specificity for the sequence (Asp)(4)-Lys. It converts trypsinogen to trypsin. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for in vitro cleavage of fusion proteins. cDNA encoding the catalytic chain of Chinese bovine enterokinase was cloned and its encoding amino acid sequence is identical to the previously reported sequence although there are two one-base mutations which do not change the encoded amino acid. The EK catalytic subunit cDNA was cloned into plasmid pET32a, and fused downstream to the fusion partner thioredoxin (Trx) and the following DDDDK enterokinase recognition sequence. The recombinant bovine enterokinase catalytic subunit was expressed in Escherichia coli BL21(DE3), and most products existed in soluble form. After an in vivo autocatalytic cleavage of the recombinant Trx-EK catalytic domain fusion protein, intact, biologically active EK catalytic subunit was released from the fusion protein. The recombinant intact EK catalytic subunit was purified to homogeneity with a specific activity of 720 AUs/mg protein through ammonium sulfate precipitation, DEAE chromatography, and gel filtration. The purified intact EK catalytic subunit has a K(m) of 0.17 mM, and K(cat) is 20.8s(-1). From 100 ml flask culture, 4.3 mg pure active EK catalytic subunits were obtained.
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PMID:Expression, purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli. 1213 63

The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.
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PMID:Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae. 1216 6

Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 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. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.
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PMID:Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli. 1296 50

Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.
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PMID:[Expression of neurotoxin II from Naja oxiana cobra venom in Escherichia coli in a hybrid form with thioredoxin]. 1504 Mar 1

Puroindolines (PIN) are low molecular weight, cysteine-rich, endosperm-specific, basic proteins with a unique tryptophan-rich domain found in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) as well as other members of Triticaceae. PINs appear to be involved in both flour softness as well as resistance against fungal diseases. These proteins are known to be the major components of 'friabilin' associated with the surface of water washed starch grains and possess lipid binding properties. Structural characterization of puroindolines from Triticum monococum was initiated by amplifying and subsequently cloning the corresponding pin gene into an expression vector, known as pET-32a(+). The protein contains five tryptophanin domains and ten cysteine residues. The pinB gene was fused with the 109aa Trx.Tag thioredoxin for a high-level expression. The cloning sites used for producing fusion proteins also contained cleavable His.Tag and S.tag sequences for detection and purification. After transformation of competent Origami cells, fusion protein expression was detected by growing a transformant in LB medium in the presence of 0.1 mM IPTG at room temperature for 6 hrs on a shaker. Both soluble and insoluble fusion proteins were extracted from Origami cells after sonication. Ni-NTA column (Qiagen) was used to extract and purify these fractions. Following an overnight digestion of the recombinant protein with enterokinase at room temperature, the corresponding fractions were electrophoresed in polyacrylamide gel, electroblotted onto a nitrocellulose membrane and cross-reacted with the anti-friabilin monoclonal antibody. We found that the recombinant PINB protein had a molecular weight of 16 kDa whereas TrxB was 21 kDa. Fusion protein ran at 34 kDa. PINB protein from wheat was shown to be immunologically related to a homologue, tryptophanin, in oat seed. Further study is currently underway to characterize these proteins structurally using NMR.
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PMID:Cloning and expression of pinB gene from Triticum monococum seeds. 1515 Dec 87

The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 +/- 1.1) x 10(-11) M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in investigations where the protein dimerization is undesirable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
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PMID:[I87E mutation prevents barstar dimerization]. 1558 16

C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited internally replacing the DDDDK in another construct of thioredoxin/mouse NT-proCNP, it was found to be poorly processed by enterokinase. Our results showed that non-target sequences can be preferentially recognized over the canonical DDDDK sequence when located accessibly at the ends of proteins.
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PMID:Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites. 1586 19

The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.
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PMID:Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity. 1587 1

The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.
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PMID:Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin. 1621 69

Scytovirin (SVN) is a novel anti-human immunodeficiency virus (HIV) protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds. A synthetic gene that encodes scytovirin was constructed, and expressed in Escherichia coli, with thioredoxin (TRX) fused to its N-terminus (TRX-SVN). Most of the expressed protein was in soluble form, which was purified by a polyhistidine tag affinity purification step. SVN was then cleaved from TRX with enterokinase and separated from the TRX partner by C18 reversed-phase HPLC. This production method has proven superior to earlier synthetic attempts and recombinant procedures using a standard expression system. The current system resulted in yields of 5-10mg/L of purified SVN for structural studies and for preclinical development of SVN as a topical microbicide for HIV prophylaxis.
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PMID:Overexpression and purification of scytovirin, a potent, novel anti-HIV protein from the cultured cyanobacterium Scytonema varium. 1628 3


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