Glutathione S-Transferase/ras Fusion Protein: A Tool for Affinity Chromatography of ras Associated Proteins

ras Oncogenes ◽  
1989 ◽  
pp. 255-260 ◽  
Author(s):  
Hiroshi Maruta
Keyword(s):  
Fusion Protein ◽  
Affinity Chromatography ◽  
Protein A ◽  
Glutathione S Transferase ◽  
Associated Proteins

scholarly journals Positive Selection of Specific Antibodies Produced against Fusion Proteins

2020 ◽  
Vol 3 (2) ◽  
pp. 37
Author(s):  
Lukas Kramberger-Kaplan ◽  
Tina Austerlitz ◽  
Holger Bohlmann
Keyword(s):  
Fusion Protein ◽  
Affinity Chromatography ◽  
Positive Selection ◽  
Fusion Proteins ◽  
Protein A ◽  
High Specificity ◽  
Western Blots ◽  
Specific Antibodies ◽  
E Coli ◽  
Selection Of

A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

Cross-species reactivity of a monoclonal antibody against glutathione S-transferase fusion protein of human β2-adrenergic receptor

IUBMB Life ◽  
1998 ◽  
Vol 45 (2) ◽  
pp. 215-225
Author(s):  
Chan Young Shin ◽  
Suk-jo Kang ◽  
Mi-ryoung Song ◽  
Kyu Hwan Park ◽  
Dong Ook Seo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Adrenergic Receptor ◽  
Glutathione S Transferase ◽  
Β2 Adrenergic Receptor

scholarly journals Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

1980 ◽  
Vol 191 (3) ◽  
pp. 799-809 ◽  
Author(s):  
R G Sutcliffe ◽  
B M Kukulska-Langlands ◽  
J R Coggins ◽  
J B Hunter ◽  
C H Gore
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Carbohydrate Content ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger

1998 ◽  
Vol 76 (5) ◽  
pp. 837-842 ◽  
Author(s):  
Daniel Gebreselassie ◽  
Krishna Rajarathnam ◽  
Larry Fliegel
Keyword(s):  
Circular Dichroism ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Cytoplasmic Domain ◽  
Random Coil ◽  
Glutathione S Transferase ◽  
Carboxyl Terminal ◽  
Circular Dichroïsm

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.Key words: Na+/H+ exchanger, pH regulation, membrane protein, circular dichroism.

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