共查询到20条相似文献,搜索用时 0 毫秒
1.
2.
3.
Palombo G De Falco S Tortora M Cassani G Fassina G 《Journal of molecular recognition : JMR》1998,11(1-6):243-246
We reported previously that TG19318, a synthetic ligand deduced from the screening of combinatorial libraries, displays specific and selective recognition properties for immunoglobulins of the G class and can be used conveniently for affinity chromatography purification of monoclonal and polyclonal antibodies. In this study we have extended the ligand characterization, examining its ability to bind IgA from cell culture supernatants and from IgG-deprived serum. Affinity columns prepared by immobilizing TG19318 on Sepharose allowed convenient one-step purification of monoclonal IgA directly from crude feedstocks, in high yield and with full recovery of immunoreactivity. Optimal column adsorption occurred with phosphate buffer at neutral pH, while elution of adsorbed IgA could be accomplished by a buffer pH change to acidic or basic conditions. Column capacity was close to 7 mg IgA/ml support. 相似文献
4.
Farnesyl pyrophosphate synthetase (EC 2.5.1.1) from chicken liver, pig liver, and yeast has been purified to homogeneity in a single chromatographic step by affinity chromatography. The affinity ligand, geranylmethylphosphonophosphate, is linked to Affi-Gel 10 through the phosphonophosphate moiety. The affinity gel is stable chemically and the internal phosphonophosphate linkage is not hydrolyzed by nonspecific phosphatases. A single column has been used repeatedly for over a year with no degradation in its performance. A typical purification only requires 2 days and gives a 500- to 600-fold purification of enzyme from a crude ammonium sulfate precipitate. 相似文献
5.
An affinity column was synthesized and utilized to partially purify glycerolphosphate dehydrogenase (L-glycerol-3-phosphate: NAD+ oxidoreductase, E.C.1.1.1.8) from rat skeletal muscle. The novelty of the column resides in the fact that the ligand used, 6-phosphogluconic acid, is neither an inhibitor nor a substrate of the enzyme when free in solution but when immobilized on an agarose matrix, glycerol phosphate dehydrogenase binds to it with a high degree of specificity. The bound enzyme could be eluted by either increasing the ionic strength or by addition of its natural substrate, α-glycerol phosphate. Using a combination of these methods and ammonium sulfate precipitation GPDH was purified about 250 fold with a 75% yield within 24 hours. 相似文献
6.
Sanjay Adhikari 《Analytical biochemistry》2010,400(2):203-206
Protein purification is still very empirical, and a unified method for purifying proteins without an affinity tag is not available yet. In the postgenomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied for purifying any recombinant basic protein from Escherichia coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI−1 ? 6.94) may be purified from E. coli in a single step using a cation-exchanger resin, SP-Sepharose, and a selected buffer pH, depending on the pI of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step. 相似文献
7.
P V Kuznetsov O A Demin V Ia Kononenko S F Zinchuk 《Ukrainski? biokhimicheski? zhurnal》1992,64(3):20-28
Steroid-binding proteins of human blood are extracted and investigated on corticosteroid-hydrazide-oxypropyl-adsorbents by the affinity chromatography. The role of structural changes in the matrix-spacer-ligand system for different types of adsorbents is studied as well as a dependence of sorption on pH and salt environment. A dependence of the protein sorption on the isolated hydrazide groups is revealed. It is shown that hydrocortisone displays the highest binding activity to the protein pool in the affinity electroimmunoassay while the substance S-Reichstein and its 16-alpha-methyl analog--the least one. 相似文献
8.
A subclass of glutathione S-transferases as intracellular high-capacity and high-affinity steroid-binding proteins. 总被引:2,自引:1,他引:2
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The distribution of glucocorticoids incubated with rat liver cytosol preparations or administered in vivo to adrenalectomized rats was analysed by chromatographic procedures. Corticosterone or dexamethasone was co-eluted with Yb-type GSH S-transferases in anion-exchange and gel-permeation chromatography systems, and these glucocorticoids also were bound to Yb forms in analyses by immunoadsorbent and lysyl-GSH affinity matrices. Pretreatment of cytosol with lysyl-GSH to extract GSH S-transferases or incubation with excess bilirubin, which is expected to compete with steroids for binding to the protein, yielded preparations that were devoid of this major steroid-binding component. In mixtures of the multiple rat GSH S-transferases, corticosterone preferentially interacted with Yb forms rather than Ya and Yc subgroups. All of the multiple Yb forms resolved by chromatofocusing procedures retained the steroid-binding capacity. It is suggested that these abundant proteins can account for a considerable share of intracellular glucocorticoid binding and represent a high-affinity non-saturable binding component with potential to function in steroid-hormone metabolism and action. 相似文献
9.
We evaluated several gel filtration materials for suitability in spun column chromatography a rapid desalting method. The materials were evaluated for resistance to column cracking and to radial shrinkage for sample recovery and for salt separation. A series of graphs can be derived from these evaluations which relates the gel heights to sample volumes for maximum recovery and salt separation while maintaining low sample dilution. 相似文献
10.
Post-translational protein modification by the covalent conjugation of ubiquitin, originally implicated as a signal for proteolytic degradation by 26S proteasome, has now been realised to play important roles in the regulation of almost all biological processes in eukaryotes. In order to understand these processes in greater detail there is a requirement for techniques that can purify mixtures of ubiquitin-conjugated proteins, as a prerequisite to their identification and characterisation. Here we review the methods that have been applied to the bulk purification of ubiquitinated proteins and discuss their applications in proteomic analyses of the 'ubiquitome'. 相似文献
11.
12.
A purification procedure for the sex steroid-binding protein of human serum is described. The procedure is significantly superior to that recently published (K. E. Mickelson, D. C. Teller, and P. H. Pétra, 1978, Biochemistry17, 1409–1415) and should replace it for the routine preparation of homogeneous protein in relatively larger quantities. The steps involved diethylaminoethyl-cellulose chromatography, affinity chromatography on 5α-dihydrotestosterone-17α-hexanyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The most important difference between this new procedure and that previously published is the affinity adsorbent with contains the steroid covalently linked at the 17α-position rather than the 17β-position. This modification allows the purification of at least 12 mg of homogeneous protein per preparation with a 63% total yield. The properties of the homogeneous protein are the same as previously described. 相似文献
13.
A new method for purification of staphylocoagulase by a bovine prothrombin-Sepharose column 总被引:2,自引:0,他引:2
Staphylocoagulase was isolated from a culture filtrate of Staphylococcus aureus, strain st-213, by a two step purification procedure of chromatography on a bovine prothrombin-Sepharose 4B affinity column and gel filtration on Sephadex G-25. The yield of the coagulase activity ranged from 75--83% and the purified preparation gave a single precipitin line in immunodiffusion tests against anti-crude and anti-purified staphylocoagulase sera. However, the final product was shown to contain one major and two minor components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chemical analysis of the material indicated that it does not contain any cystine residues and that its NH2-terminal residue is a single isoleucine. 相似文献
14.
Daxecker H Raab M Bernard E Devocelle M Treumann A Moran N 《Analytical biochemistry》2008,374(1):203-212
To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR. 相似文献
15.
Engineering proteins for purification. 总被引:5,自引:0,他引:5
H M Sassenfeld 《Trends in biotechnology》1990,8(4):88-93
Over the past decade, a new protein purification technique has emerged as a result of recombinant DNA technology. DNA, encoding additional polypeptide or protein tags, is fused to the gene of interest. Expression of these gene fusions results in protein fusions which may be purified by techniques using the properties of the additional polypeptide tag. This has eliminated the need for extensive screening and optimization procedures previously required for purification. 相似文献
16.
We describe a technique for a rapid and efficient isolation and purification of proteins binding to defined DNA sequences. Cloned double-stranded DNA was covalently coupled to m-aminobenzyloximethylcellulose in order to purify proteins which recognize and bind to specific sequences on the DNA. The purification of two DNA-binding proteins from Drosophila melanogaster is demonstrated using the respective cloned DNA sequences. 相似文献
17.
Zawilak-Pawlik AM Kois A Zakrzewska-Czerwinska J 《Protein expression and purification》2006,48(1):126-133
An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins. 相似文献
18.
A calcium-dependent antibody for identification and purification of recombinant proteins 总被引:16,自引:0,他引:16
We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in a single chromatographic step. The marker peptide could subsequently be removed by proteolysis with the enzyme enterokinase. 相似文献
19.
A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an 3H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available. 相似文献
20.
Ikonomopoulou MP Bradley AJ Whittier JM Ibrahim K 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2006,176(8):775-782
We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K
a = 1.49 ± 0.09 × 109 M−1; 0.17 ± 0.02 × 107 M−1) and low binding capacity (B
max = 3.24 ± 0.84 × 10−5 M; 0.33 ± 0.06 × 10−4 M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier. 相似文献