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1.
Bovine aortic endothelial cell transglutaminase. Enzyme characterization and regulation of activity. 总被引:5,自引:0,他引:5
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Bovine aortic endothelial cells contain Ca2+-dependent tissue-type transglutaminase. Its activity in these cells was high, with apparent Km and Vmax. values with respect to putrescine of 0.203 mM and 18.5 nmol/min per mg of protein, and its activity was inhibited by the three competitive inhibitors dansylcadaverine, spermine and methylamine. The molecular mass of endothelial cell transglutaminase estimated by gel filtration chromatography was 88 kDa and it was immunoprecipitated by rabbit monospecific antiserum raised against rat liver transglutaminase. Its enzymic activity rose when the cell cultures reached confluence, and was further increased when their proliferation was arrested (synchronized at G0/G1 phase). Most of the enzymic activity was found in the 15,000 g soluble fraction, with only 4-22% of the activity found in the particulate fraction, depending on the state of cell proliferation. Examination of these cellular fractions by SDS/polyacrylamide-gel electrophoresis and immunoblotting revealed that at confluence endothelial cells have accumulated transglutaminase antigen in their 15,000 g particulate fraction. A series of experiments demonstrated the existence of a latent transglutaminase form in non-proliferating cells, and suggested that this might involve the formation of an inhibitory complex. Treatment of cell lysates and the 15,000 g particulate fraction with high salt concentration showed a significant increase in transglutaminase activity. Mixing experiments using the 100,000 g particulate fraction or purified rat liver transglutaminase on one hand and the cytosolic fraction on the other showed dose-dependent inhibition of the transglutaminase activity of the latter. It is concluded that endothelial cells contain a particulate fraction-residing inhibitor of transglutaminase which interacts via ionic interaction with the enzyme. 相似文献
2.
Tissue transglutaminase (E.C.2.3.2.13, R-glutaminyl-peptide: amine glutaminyl transferase), was purified from extracts of rat liver by calcium dependent affinity chromatography on casein-Sepharose. In the presence of 5 mM calcium the enzyme binds to casein Sepharose and is subsequently eluted with 5 mM EGTA. The enzyme has a molecular weight of 83,000 and its activity is dependent on calcium and reduced sulfhydryl residues. A widely distributed calcium-dependent protease (E.C. 3.4.22.17) copurified with transglutaminase by gel filtration and ion exchange chromatography. The separation of these activities prior to chromatography on casein-Sepharose is essential for the isolation of a stable transglutaminase by calcium-dependent affinity chromatography. Affinity chromatography using casein-Sepharose or other immobilized substrates may allow the calcium-dependent purification of a variety of transglutaminases. 相似文献
3.
O'Neill GM Prasanna Murthy SN Lorand L Khanna R Liu SC Hanspal M Hanada T Chishti AH 《Biochemical and biophysical research communications》2003,307(2):327-331
Intracellular transglutaminases (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, mu-calpain. Using mu-calpain null (Capn1(-/-)) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of mu-calpain. Also, the absence of mu-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of mu-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells. 相似文献
4.
Separation by anion exchange chromatography of detergent extracts from a poorly metastatic HSV-2-induced hamster fibrosarcoma, its highly metastatic variant and a highly metastatic rat fibrosarcoma indicated the presence of an inactive form of transglutaminase antigen, when eluent fractions were assayed for transglutaminase activity and antigen. This inactive antigenic transglutaminase was clearly separable from the particulate and cytosolic forms of the transglutaminase enzyme. Unlike tumours, its presence could not be demonstrated in extracts from normal rat liver. Measurement of activity levels during tumour growth indicated that the progression of the two highly metastatic tumours was accompanied by a decrease in cytosolic transglutaminase activity, whilst the activity of this enzyme form remained constant in the poorly metastatic tumour. Measurement of antigen levels indicated an inverse relationship between the level of inactive transglutaminase and the level of cytosolic transglutaminase activity, suggesting that the two forms are inter-related. Gel filtration indicated the molecular weight of the inactive form to be greater than both the particulate and cytosolic forms, and it was estimated to be 120,000. Partial proteolysis of the semi-purified inactive form, by either trypsin or thrombin, led to its activation and to the appearance of a transglutaminase similar in molecular weight and ionic mobility, both by anion-exchange chromatography and electrophoresis, to the cytosolic transglutaminase. 相似文献
5.
The purpose of this study was to investigate the implication of transglutaminases in the biology of articular chondrocytes. Transglutaminase activity measurements performed on cell lysates showed that a transglutaminase was present in chondrocytes in primary culture and that it was strongly activated by limited proteolysis. In chondrocytes dedifferentiated by subculture or retinoic acid treatment, this transglutaminase appeared to be downregulated, while type II transglutaminase expression was induced. However, protein levels, mRNA steady-state levels or transglutaminase activity in whole-cell lysates do not necessarily reflect the activity present in living cells, as it is strongly regulated. Therefore, Fluoresceincadaverine, a fluorescent polyamine, was used for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells. After incubation of chondrocytes with Fluoresceincadaverine, dedifferentiated cells exhibited an extracellula r labelling, while chondrocytes in primary culture did not, unless thrombin was added to the culture medium. In contrast, Fluoresceincadaverine labelling was not detected in the cytosol, although the transglutaminases were also partly cytosolic. By confocal microscopy and Western blot analysis of labelled cells in culture, fibronectin was shown to be the main substrate for both transglutaminases. The transglutaminases present in articular chondrocytes may, therefore, contribute to the organization and the stabilization of their extracellular matrix. 相似文献
6.
Expression of the cytosolic and particulate forms of transglutaminase during chemically induced rat liver carcinogenesis 总被引:1,自引:0,他引:1
Transglutaminase (EC 2.3.2.13) activity in chemically induced rat hepatocellular carcinomas was reduced by some 65% when compared to normal rat livers. The majority of the remaining activity (approx. 85%) was found in the particulate fraction. The use of non-ionic detergent to extract the transglutaminase activity present in both normal and tumour tissue followed by its separation on a Mono-Q column revealed two distinct peaks of activity. These peaks of activity were equivalent to those previously identified as a membrane-bound transglutaminase and the more characteristic cytosolic or tissue transglutaminase. The ratio of the activity of the cytosolic enzyme to that of the membrane-bound enzyme in normal liver was calculated as 5:1. In hepatocellular carcinomas, this ratio was reduced to 0.4:1. No significant change in the activity of the membrane-bound enzyme was detectable in tumour tissue. Comparison of the cytosolic enzyme found in hepatocellular carcinomas with that found in normal liver indicated no change in its molecular weight, Km,app for putrescine incorporation into N,N'-dimethylcasein and sensitivity to activation by Ca2+. These observations suggest that the reduction in transglutaminase activity observed in the hepatocellular carcinoma is due to a selective reduction in the expression of the cytosolic transglutaminase. 相似文献
7.
C Esposito I Caputo P Colicchio R Porta R Ragone 《Archives of biochemistry and biophysics》1999,366(1):47-54
The transglutaminase from rat coagulating gland secretion has been proposed as a new member of the transglutaminase family. Its basal activity is about 11-fold lower than those of other transglutaminases (e.g., the cytosolic tissue transglutaminase), but reaches levels comparable to those of other transglutaminases on addition of specific surfactant agents. There is no study devoted to understanding the molecular basis of this apparently anomalous activation, which is maximal at approximately 1.5 mM sodium dodecyl sulfate. We provide evidence that in the presence of this detergent modifications of the intrinsic fluorescence as well as energy transfer of the protein fluorescence to a micellar probe parallel the activation of the enzyme. As the sodium dodecyl sulfate concentration inducing maximal activation equals the critical micellar concentration, the biological activity of this transglutaminase appears to be modulated by the binding of micellar aggregates. In fact, the enzyme is modified by posttranslational modifications consisting of some lipid tails. At least two of these tails could act as aggregation nuclei of the enzyme with detergents. This behavior is different from that typical of molecular forms purified from other sources. 相似文献
8.
A transglutaminase immunologically related to tissue transglutaminase catalyzes cross-linking of cell wall proteins in Chlamydomonas reinhardtii
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The addition of primary amines to the growth medium of the unicellular green alga Chlamydomonas reinhardtii disrupts cell wall assembly in both vegetative and zygotic cells. Primary amines are competitive inhibitors of the protein-cross-linking activity of transglutaminases. Two independent assays for transglutaminase confirmed a burst of extracellular activity during the early stages of cell wall formation in both vegetative cells and zygotes. When non-inhibiting levels of a radioactive primary amine ((14)C-putrescine) were added to the growth medium, both cell types were labeled in a reaction catalyzed by extracellular transglutaminase. The radioactive label was found specifically in the cell wall proteins of both cell types, and acid hydrolysis of the labeled material released unmodified (14)C-putrescine. Western blots of the proteins secreted at the times of maximal transglutaminase activity in both cell types revealed a single highly cross-reactive 72-kD band when screened with antibodies to guinea pig tissue transglutaminase. Furthermore, the proteins immunoprecipitated by this antiserum in vivo exhibited transglutaminase activity. We propose that this transglutaminase is responsible for an early cell wall protein cross-linking event that temporally precedes the oxidative cross-linking mediated by extracellular peroxidases. 相似文献
9.
Formation of cross-linking between proteins via a gamma-glutamyl-epsilon-lysine residue is an important process in many biological phenomena including apoptosis. Formation of this linkage is catalyzed by the enzyme transglutaminase, which is widely distributed from bacteria to the animal kingdom. The simple multi-cellular organism Caenorhabditis elegans also possesses transglutaminase activity associated with apoptosis [Madi, A. et al. (1998) Eur. J. Biochem. 253, 583-590], but no gene with significant homology to vertebrate or bacterial transglutaminases has been found in the C. elegans genome sequence database. On the other hand, protein disulfide isomerases were recently recognized as a new family of transglutaminases [Chandrashekar, R. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 531-536]. To identify the molecule with transglutaminase activity in C. elegans, we isolated from C. elegans a gene homologous to ERp57, which encodes a protein disulfide isomerase, expressed it in recombinant form, and characterized the transglutaminase and protein disulfide isomerase activities of the resultant protein. The C. elegans ERp57 protein had both enzyme activities, and the transglutaminase activity had similar characteristics to the activity in lysate of the whole worm. These results suggested that the ERp57 homologue was one of the substances with transglutaminase activity in C. elegans. 相似文献
10.
Ikura K Otomo C Natsuka S Ichikawa A Wakamatsu K Ito S Taoguchi S 《Bioscience, biotechnology, and biochemistry》2002,66(6):1412-1414
Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. Previously, we found a high molecular mass transglutaminase-inhibitory substance produced by Streptomyces lavendulae Y-200 that appeared to be a melanin substance. Here, we report that synthetic tyrosine melanin inhibited various types of transglutaminases. Tyrosine melanin inhibited tissue-type transglutaminase in a competitive manner with a glutamine substrate, and also inhibited the cross-linking of casein catalyzed by a tissue-type transglutaminase. The melanized hemolymph of the silkworm and melanin solutions prepared from melanin precursors inhibited tissue-type transglutaminase. These results suggested that the melanin substances generally inhibit transglutaminases. 相似文献
11.
Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane Anchorage region 总被引:5,自引:0,他引:5
The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of [3H] acetic, [3H]myristic, or [3H]palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH2 or COOH terminus. 相似文献
12.
Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation 总被引:26,自引:0,他引:26
The predominant form of the cross-linking enzyme, transglutaminase, in cultured normal human epidermal keratinocytes, is found in cell particulate material and can be solubilized by nonionic detergent. It elutes as a single peak upon either anion-exchange or gel-filtration chromatography. Monoclonal antibodies raised to the particulate enzyme cross-react with one of two transglutaminases in the cell cytosol. The second cytosolic transglutaminase, which has distinct kinetic and physical properties from the first, does not cross-react and is not essential for formation of the keratinocyte cross-linked envelope in vitro. The anti-transglutaminase antibodies stain the more differentiated layers of epidermis in a pattern similar to that given by anti-involucrin antiserum. These observations support the hypothesis that the transglutaminase so identified is involved in cross-linked envelope formation in vivo. 相似文献
13.
Ikura K Minami K Otomo C Hashimoto H Natsuka S Oda K Nakanishi K 《Bioscience, biotechnology, and biochemistry》2000,64(1):116-124
Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 10(4) and 10(5) from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems. 相似文献
14.
Thomas Schenker Beat Trueb 《Apoptosis : an international journal on programmed cell death》1996,1(2):126-130
During our search for novel transformation-sensitive proteins whose synthesis is abolished in tumour cells we found a cDNA
clone coding for tissue transglutaminase. This enzyme was identified, at the protein as well as the mRNA level, in normal
human fibroblasts, but was completely missing in their matched SV40 transformed counterparts. Since tissue transglutaminase
has been implicated in cell cycle regulation and apoptosis, we investigated the possibility of whether this enzyme might represent
a negative marker for tumour cells. We found that its synthesis varied largely among 10 cell lines derived from spontaneous
mesenchymal tumours. While cells from a rhabdomyosarcoma and a chondrosarcoma did not produce it at all, an extremely high
expression was observed in cells from an osteosarcoma and a liposarcoma. Thus, tissue transglutaminase is not a tumour-related
marker.
This study was supported by grants from the ETH Zurich (0-20-854-94) and from the Swiss National Science Foundation (31-40337.94). 相似文献
15.
Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids 总被引:4,自引:0,他引:4
Rabbit tracheal epithelial cells undergo terminal cell division, start to express a squamous phenotype, and form cross-linked envelopes when reaching the plateau phase of the growth curve. This terminal differentiation is accompanied by a 20-30-fold increase in the activity of the cross-linking enzyme transglutaminase. This activity is found almost solely in the particulate fraction of homogenized cells and can be solubilized by nonionic detergents. This transglutaminase crossreacts with a monoclonal antibody raised against type I transglutaminase, but does not react with an antiserum against type II transglutaminase. The tracheal transglutaminase contains a protein subunit of approximately 92 kDa. The omission of epidermal growth factor from the medium or the addition of fetal bovine serum, conditions that induce terminal cell division and expression of a squamous phenotype, enhance transglutaminase activity. High calcium concentrations only stimulate transglutaminase activity after the cells become committed to terminal cell division. Retinoids, which inhibit the expression of the squamous phenotype but not terminal cell division, inhibit the enhancement in transglutaminase activity induced by either confluency or serum, indicating that this enzyme activity is under the control of retinoids. Some retinoids are active at concentrations as low as 10(-12) M. The ability of retinoids to inhibit transglutaminase activity correlates well with their capacity to bind to the retinoic acid-binding protein. Our results show that the increase in transglutaminase activity correlates with the induction of the terminal differentiated phenotype and suggest that this enzyme can function as a marker for this program of differentiation of rabbit tracheal epithelial cells in culture. Our results identify the transglutaminase as type I transglutaminase and are in agreement with the concept that this transglutaminase is involved in the formation of cross-linked envelopes. 相似文献
16.
Subcellular localization of a membrane-associated transglutaminase activity in rat liver 总被引:3,自引:0,他引:3
C W Slife M D Dorsett G T Bouquett A Register E Taylor S Conroy 《Archives of biochemistry and biophysics》1985,241(2):329-336
Fractionation of rat liver by homogenization and differential centrifugation revealed that only about 83% of the transglutaminase activity in the tissue is in a soluble form, and that the remainder is associated with the particulate fraction. This latter activity remained with the membranes even after they were extensively washed to remove 99% of such soluble enzymes as lactate dehydrogenase and aldolase. Subsequent fractionation of the membranes by isopycnic density gradient centrifugation in sucrose resulted in a single band of transglutaminase activity at a density of 1.194 g/cm3. This activity was coincident with the major band of plasma membranes, which was identified by its content of 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and leucine aminopeptidase activities. After treatment with digitonin and fractionation on sucrose gradients, the transglutaminase activity and the plasma membrane marker enzyme activities were found at a new density of 1.210 g/cm3, while the enzyme markers for the other membrane fractions remained unchanged. From these data, we conclude that approximately 17% of the transglutaminase activity in rat liver is specifically associated with the plasma membranes. 相似文献
17.
H C Kim W W Idler I G Kim J H Han S I Chung P M Steinert 《The Journal of biological chemistry》1991,266(1):536-539
In order to study the expression and role of transglutaminases in the formation of the cross-linked cell envelope of human epidermis, we have used a synthetic oligonucleotide encoding the consensual active site sequence of known transglutaminase sequences. By Northern blot analysis, newborn foreskin epidermis expresses three different mRNA species of about 3.7, 3.3, and 2.9 kilobases while normal cultured epidermal keratinocytes express only the 3.7- and 2.9-kilobase species. The largest species corresponds to a known ubiquitous tissue type II or transglutaminase C activity, the smallest corresponds to a known type I or transglutaminase K activity, and the mid-sized component apparently encodes a transglutaminase E activity that has recently been shown to be expressed in terminally differentiating epidermis (Kim, H. C., Lewis, M. S., Gorman, J. L., Park, S. C., Girard, J. E., Folk, J. E. & Chung, S. I. (1990) J. Biol. Chem., in press). Using the active site oligonucleotide as a probe, we have isolated and sequenced cDNA clones encoding the transglutaminase K enzyme. The deduced complete protein sequence has 813-amino acid residues of 89.3 kDa, has a pl of 5.7, and is likely to be an essentially globular protein, which are properties expected from the partially purified enzyme. It shares 49-53% sequence homology with the other transglutaminases of known sequence, especially in regions carboxyl-terminal to the active site, and possesses sequences likely to confer its Ca2+ dependence. Interestingly, its larger size is due to extended sequences on its amino and carboxyl termini, absent on the other transglutaminases, that may define its unique properties. 相似文献
18.
Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region. 总被引:3,自引:0,他引:3
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The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation. 相似文献
19.
Malika Lajemi SYLVIE Demignot Laurence Borge Sophie Thenet-Gauci Monique Adolphe 《Journal of molecular histology》1997,29(8):593-606
The use of Fluoresceincadaverine as a primary amine donor for detecting the endogenous substrates for active transglutaminase
in living cells was studied. Fluoresceincadaverine was found to be suitable for labelling cells in culture as it did not induce
cytotoxicity when used at 0.5 mSmD in culture media and diffused throughout the cell. After appropriate fixation using methanol,
Fluoresceincadaverine-labelled cells were observed by direct fluorescence microscopy, allowing visualization of the substrates
for active transglutaminase. Simultaneous detection of transglutaminase and of Fluoresceincadaverine incorporated into proteins
strongly suggested that cytosolic transglutaminase was inactive in these living cells. However, transglutaminase co- distributed
with Fluoresceincadaverine-labelled structures, which resembled a lattice. Fluoresceincadaverine-labelled proteins detected
by Western blotting using an anti-Fluorescein antibody showed that, in living cells, the major transglutaminase substrate
migrated at an apparent molecular weight of 220 kDa, as does fibronectin. Fibronectin was found to co-distribute with Fluoresceincadaverine-labelled
lattice. This confirmed that these lattice structures were extracellular and, therefore, that transglutaminase is in an active
form in this compartment. This opportunity to perform morphological and biochemical analyses in the search for transglutaminase
substrates in living cells should help in determining the specific function of transglutaminases in a particular cell type
as well as in universal cellular events, such as apoptosis or cell growth.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
20.
Recent evidence suggests that aberrant transglutaminase activity is associated with a wide variety of diseases. Tissue transglutaminase is the most widely distributed of the six well-characterized transglutaminases in humans. We describe a method for expressing hexahistidine-tagged human tissue transglutaminase in Escherichia coli BL21(DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with CelLytic B Bacterial Cell Lysis/Extraction Reagent was accomplished by immobilized metal (Ni2+) affinity column chromatography. The procedure typically yields highly purified and highly active recombinant human tissue transglutaminase in about 1 day (about 0.6 mg/from a 1-liter culture). 相似文献