共查询到20条相似文献,搜索用时 15 毫秒
1.
Rapid partial purification of S-adenosyl-L-methionine decarboxylase by affinity chromatography 总被引:1,自引:0,他引:1
A Sepharose-ethylenediamine-PCMB column can be used to obtain a rapid purification of S-adenosyl-L-methionine decarboxylase. PCMB-affinity fractions from both rat liver and sea urchin eggs have high specific activity, particularly the latter. The activity of the purified rat liver enzyme is stimulated by the addition of either putrescine or spermidine, whereas the purified enzyme fraction from sea urchin eggs has no measurable activity without the addition of either putrescine or spermidine. In both preparations there is a stoichiometric relationship between the release of 14CO2 from S-adenosyl-L-carboxyl-14C-methionine and the formation of spermidine. 相似文献
2.
Rapid purification of calf uterine estrogen receptor (ER) to near homogeneity has been accomplished by use of sequence-specific DNA affinity resin. Very high selectivity for the estrogen receptor is achieved through the use of DNA-Sepharose containing eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-binding and DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68,000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor. 相似文献
3.
The rapid purification of dehydrogenases by a modification of affinity chromatography was investigated. A ternary complex enzyme-NAD(H)-inhibitor (E-NADH-I) was formed by the addition of coenzyme and a substrate-competitive inhibitor to the dehydrogenases initially separated from nondehydrogenases by an NAD-affinity column. The enzyme in the ternary complex cannot rebind to the NAD-agarose column in the presence of inhibitor. As all other dehydrogenases do, this yields a highly purified enzyme-inhibitor complex. Aldehyde dehydrogenases in the presence of chloral hydrate and alcohol dehydrogenase with pyrazole were purified as their E-NAD+-I ternary complexes, while lactic dehydrogenase in the presence of oxamate was purified as the E-NADH-I complex. This technique allows for the rapid separation of a specific dehydrogenase from other dehydrogenases. The technique should be applicable to the purification of other enzymes exhibiting ordered sequential binding. 相似文献
4.
Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues. 相似文献
5.
The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities. 相似文献
6.
Both plastocyanin and cytochrome f were purified using a combination of affinity chromatography together with established methods. Plastocyanin was partially purified using the method of Davis and San Pietro (Anal. Biochem. 95 (1979) 254-259), after which it was further purified using a column of cytochrome c covalently attached to Sepharose 4B. The affinity column was prepared using the method of Godinot and Gautheron (Methods Enzymol. 54 (1979) 112-114). The final purity index ratio (A278/A597) was less than 1.2, which is equal to that obtained using the more expensive FPLC procedure (Anderson, G.P., Sanderson, D.G., Lee, C.H., Durell, S., Anderson, L.B. and Gross, E.L. (1987) Biochim. Biophys. Acta 894, issue 3). Cytochrome f was partially purified using a modification of the method of Matazaki et al. (Plant Cell. Physiol. 16 (1975) 237-246) and bound to an affinity column of plastocyanin covalently attached to Sepharose 4B. Cytochrome f purified using this procedure had a purity index ratio (A554.5/A277) of 1.2. Both proteins are tyrosine proteins containing no tryptophan residues. After the affinity chromatography step, the fluorescence emission spectrum of either plastocyanin or cytochrome f was typical of a tyrosine protein free from tryptophan contamination. 相似文献
7.
8.
《Journal of biochemical and biophysical methods》1996,31(1-2):31-37
Dye-ligand chromatography has been used successfully for the purification of extracellular thermostable proteinases from thermophilic Bacillus and Thermus cultures. Single step purification factors of up to 115-fold (for Thermus protease) and 2195-fold (for Bacillus protease) were obtained. Elution studies suggested that the mode of binding involved the enzyme active sites. The method was readily scaleable to 600 l volume. 相似文献
9.
F.T Kreutz D.S Wishart M.R Suresh 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,714(2):235
Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species. 相似文献
10.
N. Elango S. Janaki A.R. Rao 《Biochemical and biophysical research communications》1978,83(4):1388-1395
The purification of Glyoxalase I from rabbit liver using Blue Dextran-Sepharose-4B and S-hexyl Glutathione Sepharose-6B is described. Elution of Glyoxalase I from both the columns was accomplished with S-hexyl glutathione, a competitive inhibitor of the enzyme. The purified enzyme gave two bands on disc electrophoresis. After treatment with glutathione, only one band was found. Except for these interconvertible forms, the purified enzyme was homogeneous as shown by disc electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 相似文献
11.
Han Y Forde GM 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,874(1-2):21-26
Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. 相似文献
12.
High-throughput protein purification using an automated set-up for high-yield affinity chromatography 总被引:2,自引:0,他引:2
One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity. 相似文献
13.
Estrogen receptor purification by affinity chromatography using an orange triazine dye 总被引:1,自引:0,他引:1
A rapid two-step procedure was devised for the purification of the estrogen receptor from the calf uterus. A 900- to 1700-fold purification of the estrogen receptor was obtained using ammonium sulfate precipitation followed by dye affinity chromatography with Reactive Orange 14 immobilized to Sepharose. The Reactive Orange 14-Sepharose was used to purify the estrogen receptor in the presence or absence of estradiol as well as to purify the progesterone receptor. The purified estrogen receptor retained its estradiol- and DNA-binding properties and sedimented into sucrose gradients as the 5 S receptor dimer. The Reactive Orange 14-Sepharose is easily prepared and offers a higher yield and purity of the estrogen receptor than that afforded by estrogen- or heparin-Sepharose chromatography. 相似文献
14.
We have used alkyl ether analogs of ethanolamine and choline phospholipids as ligands to purify phospholipase A2 (EC 3.1.1.4) from Crotalus adamanteus venom by affinity chromatography. One of the affinity columns was prepared with rac-1-(9-carboxy)nonyl-2-hexadecylglycero-3-phosphocholine linked to AH-Sepharose 4B via the carboxyl group. Specific adsorption of phospholipase A2 to this column was achieved in buffer containing Ca2+, and the enzyme was eluted in buffer containing EDTA. The two enzymes from this venom were prepared in good yield (greater than 90%), and were homogeneous as judged by polyacrylamide gel electrophoresis. Retention of phospholipase A2 did not occur when the initial irrigant was devoid of Ca2+. These results support the compulsory ordered mechanism for this enzyme proposed by Wells ((1972), Biochemistry 11, 1030-1041) on the basis of kinetic considerations. The second affinity support was prepared with 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine attached through the amine moiety to CH-Sepharose 4B. Specific adsorption of phospholipase A2 to this column did not occur. These data indicate that the phospholipid base group must be accessible to the enzyme for optimal binding, and that modifications in the alkyl side chains are more desirable when designing affinity matrices for purification of enzymes involved in phospholipid metabolism. 相似文献
15.
Single-step purification of urease by affinity chromatography 总被引:5,自引:0,他引:5
16.
The purification of papain by affinity chromatography 总被引:11,自引:0,他引:11
17.
Wang Z Hsieh TC Zhang Z Ma Y Wu JM 《Biochemical and biophysical research communications》2004,323(3):743-749
The phytochemical resveratrol (trans-3,4′,5-trihydroxystilbene) is a naturally occurring polyphenol with a plethora of health-beneficial properties, including a preventive role in cancer. We surmise that resveratrol may exert its diverse biological effects by interacting with specific target proteins, denoted RTPs. To test this possibility, resveratrol was immobilized on epoxy-activated agarose forming a resveratrol affinity column (RAC), which was used to detect and isolate RTPs. Distinct RTPs can be resolved on RAC by fractionation with increasing NaCl, followed by 1 mM ATP, and finally, with 1-2 mM resveratrol. A 22-kDa polypeptide, RTP-22, eluted with resveratrol was identified by MALDI-TOF MS and cloning/expression in Escherichia coli, as dihydronicotinamide riboside quinone reductase 2 (NQO2). The utility of RAC was additionally explored with extracts derived from different staging prostate cancer cells. NQO2 was most abundant in CWR22Rv1, a model for prostate cancer transition from androgen-dependent to the hormone-refractory state, but was marginally expressed in JCA-1 cells as representing more advanced stage prostate cancer. These results provide evidence for the existence of distinctive RTPs in mammalian cells and that RAC is a facile approach to identify and purify RTPs. 相似文献
18.
Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography 总被引:1,自引:0,他引:1
Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
The SecA protein occupies a pivotal position in the public protein export pathway inEscherichia coli. The multifunctional SecA protein recognizes cytoplasmic factors associated with export including the presecretory protein and targets the complex to the inner membrane, where it acts in the early stages of protein translocation. The ability of SecA to bind ATP was the basis for the development of a novel, rapid purification scheme involving a single chromatographic step. Affinity chromatography was carried out on Red Sepharose CL-6B. The SecA present in crude extracts ofE. coli binds strongly to this dye-ligand matrix, and active protein was purified to greater than 90% homogeneity. The protein isolated by this procedure retained the previously described ATPase and RNA-binding activities of SecA. This approach should permit the rapid purification of SecA homologs from a variety microorganisms. 相似文献
20.
Rapid affinity purification of retinal arrestin (48 kDa protein) via its light-dependent binding to phosphorylated rhodopsin 总被引:2,自引:0,他引:2
Arrestin (also named '48 kDa protein' or 'S-antigen') is a soluble protein involved in controlling light-dependent cGMP phosphodiesterase activity in retinal rods, and is also known for its ability to induce autoimmune uveitis of the eye. We report a rapid and simple purification method based on the property of arrestin to bind specifically and reversibly to illuminated and phosphorylated rhodopsin [(1984) FEBS Lett. 176, 473-478]. This method does not require column chromatography and yields about 2-4 mg purified arrestin from 15 bovine retinas. Pure arrestin can be resolved by isoelectric focusing into at least 10 distinct bands, all of which stain with a monoclonal antibody specific for S-antigen. 相似文献