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1.
The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P1,P3-triphosphate (Ap3A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His6-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a Km value for Ap3A of 0.9 μM and a kcat(monomer) value of 7.2 ± 1.6 s−1 (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap3A.  相似文献   

2.
Abend A  Garrison PN  Barnes LD  Frey PA 《Biochemistry》1999,38(12):3668-3676
Fhit is the protein product of FHIT, a candidate human tumor suppressor gene. Fhit catalyzes the hydrolysis of diadenosine triphosphate (Ap3A) to AMP and ADP. Fhit is here shown to catalyze the hydrolysis in H218O with production of adenosine 5'-[18O]phosphate and ADP, proving that the substitution of water is at Palpha and not at Pbeta. The chain fold of Fhit is similar to that of galactose-1-phosphate uridylyltransferase, which functions by a double-displacement mechanism through the formation of a covalent nucleotidyl-enzyme intermediate and overall retention of configuration at Palpha. The active site of Fhit contains a histidine motif that is reminiscent of the HPH motif in galactose-1-phosphate uridylyltransferases, in which the first histidine residue serves as the nucleophilic catalyst to which the nucleotidyl group is bonded covalently in the covalent intermediate. In this work, the Fhit-catalyzed cleavage of (RP)- and (SP)-gamma-(m-nitrobenzyl) adenosine 5'-O-1-thiotriphosphate (mNBATPalphaS) in H218O to adenosine 5'-[18O]thiophosphate is shown to proceed with overall retention of configuration at phosphorus. gamma-(m-Nitrobenzyl) adenosine 5'-O-triphosphate (mNBATP) is approximately as good a substrate for Fhit as Ap3A, and both (RP)- and (SP)-mNBATPalphaS are substrates that react at about 0.5% of the rate of Ap3A. The stereochemical evidence indicates that hydrolysis by Fhit proceeds by a double-displacement mechanism, presumably through a covalent AMP-enzyme intermediate.  相似文献   

3.
Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.  相似文献   

4.
The YUH1 gene coding for ubiquitin C-terminal hydrolase 1, a deubiquitinating enzyme, was cloned from the Saccharomyces cerevisiae genomic DNA and expressed in Escherichia coli. YUH1 was fused with the 6 histidine tag at the N-terminus (H6YUH1) or C-terminus (YUH1H6) and purified by an immobilized metal affinity chromatography with high purity. By using a fluorogenic substrate, Z-Arg-Leu-Arg-Gly-Gly-AMC, the deubiquitinating activities for H6YUH1 (1.72U/mg) and YUH1H6 (1.61U/mg) were about 18 times higher than 0.092U/mg for H6UBP1, ubiquitin specific protease 1 of S. cerevisiae containing the 6 histidine residue at the N-terminus which is normally used in protein engineering. YUH1 had the optimal temperature of 27 degrees C and acidity of pH 8.5. Analysis of thermal deactivation kinetics of H6YUH1 estimated 3.2 and 1.4h of half lives at 4 and 52 degrees C, respectively. Immobilization onto the Ni-NTA affinity resin and environmental modulation were carried out to improve the stability of YUH1. Incubation of the immobilized YUH1 in 50% glycerol solution at -20 degrees C resulted in 52% of decrease in specific activity for 7days, corresponding to a 2.7-fold increase compared with that of the free YUH1 incubated in the same solution at 4 degrees C.  相似文献   

5.
We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of approximately 17 kDa monomers. It catalyses the Mg(2+)-dependent hydrolysis of diadenosine triphosphate, Ap(3)A, to AMP+ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, epsilon-(Ap(3)A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap(3)Aase presents a native molecular mass of approximately 32 kDa and no significant differences were found in K(m) values (2 microM), activating effects by Mg(2+), Ca(2+), and Mn(2+), optimum pH (7.0-7.2) or inhibition by Zn(2+) and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap(3)Aase and Fhit protein with K(i) values in the range 20-30 nM. Both human and rat Ap(3)Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap(3)Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a approximately 17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap(3)Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit-Ap(3)Aase mediated by its interaction with suramin or related drugs.  相似文献   

6.
7.
The human tumor suppressor Fhit protein exhibits diadenosine triphosphatase activity, hydrolyzing Ap(3)A to AMP and ADP. We report that Fhit protein efficiently cleaves the fluorogenic Ap(3)A analog diethenoadenosine triphosphate giving support to establish a simple fluorimetric assay for quantification of Fhit enzyme. Fluorimetric assays were initially tested to demonstrate that diethyl pyrocarbonate and suramin inhibit Fhit enzyme.  相似文献   

8.
Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 μM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.  相似文献   

9.
Huang K  Arabshahi A  Wei Y  Frey PA 《Biochemistry》2004,43(23):7637-7642
The human fragile histidine triad protein Fhit catalyzes the Mg(2+)-dependent hydrolysis of P(1)-5'-O-adenosine-P(3)-5'-O-adenosine triphosphate, Ap(3)A, to AMP and ADP. The reaction is thought to follow a two-step mechanism, in which the complex of Ap(3)A and Mg(2+) reacts in the first step with His96 of the enzyme to form a covalent Fhit-AMP intermediate and release MgADP. In the second step, the intermediate Fhit-AMP undergoes hydrolysis to AMP and Fhit. The mechanism is inspired by the chain-fold similarities of Fhit to galactose-1-phosphate uridylyltransferase, which functions by an analogous mechanism, and the observation of overall retention in configuration at phosphorus in the action of Fhit (Abend, A., Garrison, P. N., Barnes, L. D., and Frey, P. A. (1999) Biochemistry 38, 3668-3676). Direct evidence in support of this mechanism is reported herein. Reaction of Fhit with [8,8'-(3)H]-Ap(3)A and denaturation of the enzyme in the steady state leads to protein-bound tritium corresponding to 11% of the active sites. Similar experiments with the poor substrate MgATP leads to 0.9% labeling. The mutated protein H96G-Fhit is completely inactive against MgAp(3)A. However, it is chemically rescued by free histidine. H96G-Fhit also catalyzes the hydrolysis of adenosine-5'-phosphoimidazolide, AMP-Im, and of adenosine-5'-phospho-N-methylimidazolide, AMP-N-MeIm. The hydrolyses of AMP-Im and of AMP-N-MeIm by H96G-Fhit are thought to represent chemical rescue of the covalent Fhit-AMP intermediate. Wild-type Fhit is also found to catalyze the hydrolyses of AMP-Im and of AMP-N-MeIm nearly as efficiently as the hydrolysis of MgAp(3)A. The results indicate that Mg(2+) in the reaction of Ap(3)A is required for the first step, the formation of the covalent intermediate Fhit-AMP, and not for the hydrolysis of the intermediate in the second step.  相似文献   

10.
Pronuclear rat eggs were cultured for 24 to 48 hours at 37 degrees C after storage at 2 to 6 degrees C for 0 to 216 hours in medium. Very high proportions (89 to 97%) of eggs cleaved to the two-cell stage after storage for 0 to 48 hours. The proportion, however, decreased rapidly in eggs stored for 72 hours (60%), and eggs stored for more than 120 hours cleaved poorly. However, when male and female pronuclei from stored eggs were transplanted into enucleated fresh eggs, 92 to 100% of the fused eggs with karyoplast stored for 0 to 144 hours cleaved. Although the cleavage rate was reduced to 50% when karyoplast from eggs stored for 168 hours was transplanted, this reduction was not significant. Complete loss of cleaving ability was observed in fused eggs with the karyoplast stored for 216 hours. These results clearly indicate that the pronuclei of rat eggs can be stored for a longer period than the cytoplasm at low temperatures (2 to 6 degrees C).  相似文献   

11.
Diadenosines as FHIT-ness instructors   总被引:5,自引:0,他引:5  
FHIT is a tumor suppressor gene that is frequently inactivated in human cancer. Although the Fhit protein is known to hydrolyze diadenosine triphosphate (Ap(3)A), this hydrolase activity is not required for Fhit-mediated oncosuppression. Indeed, the molecular mechanisms and the regulatory elements of Fhit oncosuppression are largely unknown. Here, we review physiological and pathological aspects of Fhit in the context of the Ap(n)A family of signaling molecules, as well as the involvement of Fhit in apoptosis and the cell cycle in cancer models. We also discuss recent findings of novel Fhit interactions that may lead to new hypotheses about biochemical mechanisms underlying the oncosuppressor activity of this gene.  相似文献   

12.
Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.  相似文献   

13.
Fhit protein is the product of the putative tumor suppressor fragile histidine triad (FHIT) gene. The way by which Fhit exerts its antitumor activity remains largely unknown, although the Fhit-Ap3A complex is believed to be the native signaling form of Fhit. Here, we have shown that Fhit protein interacts with hUbc9, a recombinant human SUMO-1 conjugating enzyme, in an adenosine(5')triphospho(5')nucleoside (Ap3N)-dependent manner. Our experiments showed that the dinucleoside polyphosphate hydrolase activity of Fhit is suppressed by interacting with hUbc9 protein. In the presence of equimolar hUbc9 the Vmax and Km activity of Fhit was decreased by 35%. Analysis of Fhit kinetics in the presence of different fixed concentrations of Ubc9 showed that Ubc9 is an uncompetitive inhibitor. Including SUMO-1 protein in the assay neither affected the Fhit activity nor modified the effect of Ubc9 on Fhit kinetics. Our data suggest that hUbc9-induced inhibition of Fhit may result in an elongation of the Fhit-Ap3A signaling complex lifetime leading to alteration of its antitumor activity.  相似文献   

14.
Truncated form of UBP1, an ubiquitin-specific protease ofSaccharomyces cerevisiae, was overexpressed inEscherichia coli. The hexahistidine residue (His6) was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were 40°C and pH 8.0, respectively.  相似文献   

15.
Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab+Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at -20 degrees C, 5 degrees C, 30 degrees C, or 40 degrees C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser-Cys-Asp-Lys-Thr-His-Thr sequence. The fragment due to cleavage of the Asp-Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.  相似文献   

16.
Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.  相似文献   

17.
The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.  相似文献   

18.
Due to the action of a serum protease, the two most cathodal isoinhibitors of the alpha 1-proteinase inhibitor (alpha 1-PI) are cleaved at the Gly5-Asp6 bond and lack two negative charges. In spite of this, these can bind trypsin and chymotrypsin, showing that the N-terminal pentapeptide is not indispensable for inhibition function. Pancreatic proteases also cleave a bond near the N-terminus in alpha 1-PI, resulting in a loss of two negative charges and a corresponding cathodal shift in the electrofocusing behavior of the isoinhibitors. Trypsin cleaves isoinhibitors near the N-terminus at a large inhibitor excess and unless an additional cleavage takes place, at least two of the new isoinhibitors remain active. An additional cleavage(s), most likely at a distance of 30-40 residues from the C-terminus results in a corresponding decrease of the molecular mass and a loss of inhibition function. Although the C-terminal cleavage peptide does separate from the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it remains associated with it under conditions of polyacrylamide gel isoelectric focusing. Chymotrypsin also cleaved alpha 1-PI near the N-terminus but this could be observed only at protease excess and the modified isoinhibitors did not form complexes with chymotrypsin. The molecular polymorphism of alpha 1-PI is partly explained by the absence of the N-terminal pentapeptide from some of the isoinhibitors.  相似文献   

19.
Using the two-hybrid technique we identified a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H+-ATPase from Arabidopsis thaliana (aa 847-949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50-70% at the aa level over stretches of 200-600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H+-ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+-ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H+-ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H+-ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.  相似文献   

20.
A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein. Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FXa cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity. The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. 1H NMR is used to analyze the N-extended BPTI analogues.  相似文献   

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