Affinity chromatography

In this study, the DNA fragment encoding for the C-terminus domain of the AopB from Aeromonas hydrophila AH-1 was cloned into pET-M expression vector and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant AopB-C-terminus domain was successfully purified using immobilized nickel affinity chromatography as a soluble form. Crosslinking analysis among AopB-C-terminus molecules in solution showed that this domain existed as a mixture of tetramer, trimer, dimer, and monomer forms.

6p guitaracoustic02 08-12-2021 18 3   Download

Fibroblast growth factor-2 (FGF-2) is a multifunctional protein that plays an important role in the regulation of proliferation, differentiation and migration of a variety of cells. The recombinant human FGF-2 (rhFGF-2) is currently used in stem cell culture, medicine and cosmetic products. In this study, we aim to produce secreted rhFGF-2 protein from a Pichia pastoris strain containing multiple copies of the fgf-2 gene to eliminate the disadvantages of intracellular expression systems

9p larachdumlanat127 02-01-2021 13 2   Download

In this study, the recombinant endoglucanase was purified by his - tag affinity chromatography with differrent processes, such as using phosphate buffer with or without sodium cloride, pretreatment of samples with ammonium sulphate before supplying into affinity column, using various concentration of imidazole for washing.

9p tamynhan10 24-10-2020 17 1   Download

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mam-malian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE....

8p tumor12 20-04-2013 39 2   Download

Increasing evidence suggests that melatonin can exert some effect at nuclear level. Previous experiments using binding techniques clearly showed the existence of specificmelatonin binding sites in cell nucleus of rat liver. To further identify these sites, nuclear extracts fromrathepatocyteswere treated withdifferent percentages of ammoniumsulfate andpurified by affinity chromatography. Subsequent ligandblot analysis shows the presence of two polypeptides of
60 and
74 kDa that bind specifically to melatonin. ...

9p tumor12 20-04-2013 57 2   Download

Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of prote-ase inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multi-valent protease inhibitor that may control the action of different proteases.

7p fptmusic 16-04-2013 40 3   Download

Arthropod cuticles play an important role as the first barrier against inva-ding pathogens. We extensively determined the sequences of horseshoe crab cuticular proteins. Proteins extracted from a part of the ventral side of the cuticle were purified by chitin-affinity chromatography, and separated by two-dimensional SDS⁄PAGE. Proteins appearing on the gel were designa-ted high molecular mass chitin-binding proteins, and these proteins were then grouped into classes based on their approximate isoelectric points and predominant amino acid compositions. ...

13p fptmusic 12-04-2013 34 2   Download

The aminocoumarin antibiotic coumermycin A1produced byStreptomyces rishiriensisDSM40489 contains two amide bonds. The biosynthetic gene cluster ofcoumermycin con-tains a putative amide synthetase gene, couL, encoding a protein of529 amino acids. CouL was overexpressed as hexahistidine fusion protein inEscherichia coliand purified by metal affinity chromatography, resulting in a nearly homogenous protein.

7p fptmusic 12-04-2013 36 4   Download

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl trans-ferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; ...

0p awards 05-04-2013 40 2   Download

Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below charac-terize the differences between the precursor and mature forms of water-melon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were puri-fied to homogeneity using Ni 2+ –NTA affinity chromatography.

0p awards 05-04-2013 45 1   Download

The extremely heat-stable 5¢-methylthioadenosine phos-phorylase from the hyperthermophilic archaeonPyrococcus furiosuswas cloned, expressed to high levels inEscherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi–Bi mechanism and phosphate binding precedes nucleo-side binding in the phosphorolytic direction....

11p awards 05-04-2013 39 2   Download

We have previously shown that high density lipoprotein is the most abundant protein in the carp plasma and dis-plays bactericidal activityin vitro. Therefore the aim of this study was to analyze the contribution of its principal apolipoproteins, apoA-I and apoA-II, in defense. Both apolipoproteins were isolated by a two step procedure involving affinity and gel filtration chromatography and were shown to display bactericidal and/or bacteriostatic activity in the micromolar range against Gram-positive and Gram-negative bacteria, including some fish patho-gens. ...

7p awards 05-04-2013 39 3   Download

Carboxypeptidases were purified fromguts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carb-oxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymesofclan MC [Barrett,A.J.,Rawlings,N. D.& Woessner, J. F. (1998)Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted spe-cificity towards C-terminal acidic residues....

12p dell39 03-04-2013 43 3   Download

TheispC gene of Arabidopsis thalianawas expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia colistrain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate at a rate of 5.6lmolÆmin )1 Æmg )1 (kcat 4.4 s )1 ). The Michaelis constants for 1-deoxy-d-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30lm, respectively. ...

13p inspiron33 25-03-2013 36 5   Download

Parkia platycephalalectin 2 was purified from Parkia platycephala(Legumi-nosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the for-mation of three intramolecular disulfide bonds.

13p inspiron33 25-03-2013 31 3   Download

Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phos-phoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best.

12p galaxyss3 21-03-2013 38 3   Download

Abrus pulchellusseeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity.

12p media19 05-03-2013 33 2   Download

After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhib-itor and by ion exchange chromatography.

16p vinaphone15 25-02-2013 35 1   Download

Dendritic cell-specific intracellular adhesion molecule-3-grabbing noninte-grin (DC-SIGN) is a member of the C-type lectin family selectively expressed on immune-related cells. In the present study, we performed a systematic interaction analysis of DC-SIGN and its related receptors, DC-SIGN-related protein (DC-SIGNR) and liver and lymph node sinusoi-dal endothelial cell C-type lectin (LSECtin) using frontal affinity chroma-tography (FAC).

17p viettel02 19-02-2013 42 3   Download

We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy. Bacterial expression can be used to express small recombi-nant antibodies on a large scale; however, their overexpression often results in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography.

11p mobifone23 18-01-2013 42 3   Download