Novel affinity chromatography method for the efficient purification of recombinant Binder of SPerm homolog proteins

2020 ◽  
Vol 43 (17) ◽  
pp. 3458-3466
Author(s):  
Samin Sabouhi Zarafshan ◽  
Puttaswamy Manjunath
Keyword(s):  
Affinity Chromatography ◽  
Chromatography Method

scholarly journals Purification of Porcine Circovirus Type 2 Using an Affinity Chromatography Based on a Neutralizing Monoclonal Antibody against Viral Capsid Protein

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1564
Author(s):  
Haiqiao Bian ◽  
Chong Yu ◽  
Yanwu Wei ◽  
Li Feng ◽  
Changming Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Affinity Chromatography ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromatography Method ◽  
Viral Adsorption

Porcine circovirus type 2 (PCV2) is a DNA virus without an envelope. The viral particle is icosahedral and has a diameter of approximately 17 nm. In order to obtain the purified virus, a broad-spectrum monoclonal antibody 3A5 against PCV2 was coupled to CNBr-activated SepharoseTM 4B, and an affinity chromatography was established for PCV2 purification. A total of 6.5 mg of purified PCV2a/LG with 97% purity was obtained from 120 mL of the viral culture medium, and only PCV2 was detected by electron microscopy. No significant changes in the antigenic characteristics of the purified virus were detected by a capture enzyme-linked immunosorbent assay (ELISA). Furthermore, the titer of the purified PCV2 was 100 times higher than that of the unpurified virus. This affinity chromatography method was also used to purify PCV2b/LN590516 and PCV2d/SD446F16, and the purified viruses were detected by electron microscopy, capture ELISA, and virus titration, respectively. The results showed that these two strains can be successfully purified, but the yield is lower than that of the PCV2a strain. In addition, the purified virus could be used to study the viral adsorption and invasion of PK15 cells using indirect immunofluorescence assays. A large number of PCV2 signals were detected to transfer from the cellular surface to the periphery of the nucleus of the PK15 cells after 30 min of adsorption of the PCV2 to the PK15 cells. The affinity chromatography is a simple and convenient tool to obtain PCV2 with high purity. It could be applied for virus structure analysis, antibody preparation, and viral adsorption and invasion research.

Investigation and validation of the affinity chromatography method for measuring glycated albumin in serum and urine

1991 ◽  
Vol 202 (1-2) ◽  
pp. 11-22 ◽  
Author(s):  
Anne C. Silver ◽  
Edmund Lamb ◽  
William R. Cattell ◽  
Anne B.St.J. Dawnay
Keyword(s):  
Affinity Chromatography ◽  
Glycated Albumin ◽  
Chromatography Method ◽  
Serum And Urine

Screening the recognition properties of peptide hormone sequence mutants by analytical high performance liquid affinity chromatography on immobilized neurophysin

1988 ◽  
Vol 53 (11) ◽  
pp. 2627-2636 ◽  
Author(s):  
Giorgio Fassina ◽  
Michal Lebl ◽  
Irwin M. Chaiken
Keyword(s):  
Affinity Chromatography ◽  
High Performance ◽  
Peptide Hormone ◽  
Protein Recognition ◽  
Affinity Matrix ◽  
Structure Modification ◽  
Binding Properties ◽  
Chromatography Method ◽  
Contact Residues ◽  
Critical Contact

Analytical high performance liquid affinity chromatography with immobilized neurophysin was used as a molecular screen to evaluate the effects of peptide hormone structure modification on protein recognition. Immobilization of neurophysin on silica and highly cross-linked agarose occurred with retention of oxytocin and vasopressin binding properties. The effects of one-residue-at-a-time mutation, multi-site sequence simplification, and sequence randomization of critical contact residues were evaluated by extent of binding of the peptides on the affinity matrix. The analytical chromatography method also was used as a stereoselective detector to identify racemic contaminants in peptide hormone preparations.

scholarly journals pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

2001 ◽  
Vol 67 (3) ◽  
pp. 1262-1267 ◽  
Author(s):  
Shuhei Fujimoto ◽  
Yasuyoshi Ike
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Enterococcus Faecalis ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Chromatography Method ◽  
Shuttle Vectors ◽  
E Coli ◽  
One Step

ABSTRACT Two novel Enterococcus faecalis-Escherichia colishuttle vectors that utilize the promoter and ribosome binding site ofbacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli andE. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coliand E. faecalis at levels of 103 and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

An Inexpensive, Rapid and Precise Affinity Chromatography Method for the Measurement of Glycosylated Haemoglobins

1983 ◽  
Vol 20 (3) ◽  
pp. 129-135 ◽  
Author(s):  
Pauline M Hall ◽  
J G H Cook ◽  
B J Gould
Keyword(s):  
Affinity Chromatography ◽  
Clinical Chemistry ◽  
Glycosylated Haemoglobin ◽  
Chemistry Laboratory ◽  
Chromatography Method ◽  
Clinical Chemistry Laboratory ◽  
Glycosylated Haemoglobins ◽  
Working Day ◽  
Immediate Analysis ◽  
The Cost

We have assessed an affinity chromatography technique, using commercially available materials, for the estimation of total glycosylated haemoglobin in the routine clinical chemistry laboratory. The method gives good discrimination between normals (7·31±0·92%) and diabetics (12·70±2·88%) and has excellent precision (CV 1·5-2·0%). Labile glycosylated haemoglobin is normally removed as it is so variable. There is no significant correlation between labile glycosylated haemoglobin and blood glucose. Immediate analysis of incubated haemolysates is preferable to storage of haemolysates or erythrocytes. The affinity gel can be reused about 16 times, but oxidation must be reduced by keeping the gel at 4°C in the dark when not in use. The cost of the gel is about 7p a test and 60 samples can be analysed in a working day. The method is not affected by the presence of up to 20% met-haemoglobin and should also give correct values for samples containing genetic variants of haemoglobin.

scholarly journals Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule

The Analyst ◽  
2016 ◽  
Vol 141 (24) ◽  
pp. 6571-6582 ◽  
Author(s):  
Nur Mustafaoglu ◽  
Tanyel Kiziltepe ◽  
Basar Bilgicer
Keyword(s):  
Affinity Chromatography ◽  
Binding Site ◽  
Small Molecule ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Chromatography Method ◽  
Antibody Purification ◽  
Membrane Affinity ◽  
Affinity Membrane ◽  
Site Targeting

m-NBST is a small-molecule based membrane affinity chromatography system that utilizes the NBS, providing high levels of antibody recovery and purity.

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method

2009 ◽  
Vol 315 (2) ◽  
pp. 176-189 ◽  
Author(s):  
Thomas C. Scanlon ◽  
Bruce Gottlieb ◽  
Thomas M. Durcan ◽  
Edward A. Fon ◽  
Lenore K. Beitel ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Interacting Proteins ◽  
Chromatography Method

scholarly journals The transport of 125I-labeled human high molecular weight urokinase across the intestinal tract in a dog model with stimulation of synthesis and/or release of plasminogen activators

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 69-75 ◽  
Author(s):  
K Sasaki ◽  
S Moriyama ◽  
Y Tanaka ◽  
H Sumi ◽  
N Toki ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Intestinal Tract ◽  
Gel Filtration ◽  
Intestinal Transport ◽  
Plasminogen Activators ◽  
Protein Fractions ◽  
Igg Antibody ◽  
Chromatography Method ◽  
Dog Model ◽  
Rabbit Igg

Abstract In an attempt to elucidate the mechanism of fibrinolytic enhancement by orally administered urokinase, studies on the intestinal transport of urokinase were carried out, using 125I-labeled human high mol wt urokinase, administered intraduodenally in the experimental dog model with a saphenous vein thrombus. Using the plasma sample obtained from blood 45 minutes after intraduodenal administration of the urokinase, protein fractions were isolated by a sequential two-step affinity chromatography method, first with [N alpha-(epsilon-aminocaproyl)-DL- homoarginine hexylester]-Sepharose followed by a specific anti-human low mol wt urokinase rabbit IgG-Sepharose (adsorbed-eluted and unadsorbed). Each of the isolated protein fractions was further purified by gel filtration on Sephacryl S-300. The proteins isolated by the two-step affinity chromatography method were transported human urokinase with radioactivity in the adsorbed-eluted fraction, and newly synthesized and/or released dog plasminogen activators, probably urokinase-type and tissue-activator type, without radioactivity. In an antibody quenching assay, dog urokinase and the immuno-affinity unadsorbed fraction were not neutralized, but the immuno-affinity adsorbed-eluted fraction was completely neutralized by the specific anti-urokinase IgG antibody. Proteins isolated from control plasma (after administration of saline) by the two-step affinity chromatography method in the unadsorbed fraction had negligible amounts of activator activity. In these studies, we were able to show that synthesis of plasminogen activators was stimulated, with the activators being released, from either the liver or the vascular endothelium. Also we showed that urokinase is transported across the intestinal tract in the dog model.

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