Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

1992 ◽  
Vol 39 (8) ◽  
pp. 828-832 ◽  
Author(s):  
John Hartman ◽  
Paru Daram ◽  
Raymond A. Frizzell ◽  
Thomas Rado ◽  
Dale J. Benos ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Affinity Purification ◽  
Glutathione S Transferase ◽  
Recombinant Fusion Proteins

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

Secretion and affinity purification of glutathione S-transferase fusion proteins from yeast

1995 ◽  
Vol 9 (11) ◽  
pp. 821-826
Author(s):  
L. A. Castelli ◽  
A. J. Petris ◽  
S. M. Carroll ◽  
I. G. Macreadie
Keyword(s):  
Fusion Proteins ◽  
Affinity Purification ◽  
Glutathione S Transferase

An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase

1992 ◽  
Vol 156 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Daniel U. Rabin ◽  
Rebecca Palmer-Crocker ◽  
Diane V. Mierz ◽  
Kwok K. Yeung
Keyword(s):  
Fusion Proteins ◽  
Glutathione S Transferase ◽  
Recombinant Fusion Proteins ◽  
Capture Assay

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

scholarly journals pC6-2/caspase-6 system to purify glutathione-S-transferase-free recombinant fusion proteins expressed in Escherichia coli

2006 ◽  
Vol 1 (4) ◽  
pp. 1820-1827 ◽  
Author(s):  
Prabhat Kumar Purbey ◽  
P Cyril Jayakumar ◽  
Milind S Patole ◽  
Sanjeev Galande
Keyword(s):  
Escherichia Coli ◽  
Fusion Proteins ◽  
Glutathione S Transferase ◽  
Recombinant Fusion Proteins ◽  
Caspase 6

Use of recombinant fusion proteins for generation and rapid characterization of monoclonal antibodies

1992 ◽  
Vol 147 (1) ◽  
pp. 1-11 ◽  
Author(s):  
D. Wunderlich ◽  
A. Lee ◽  
R.P. Fracasso ◽  
D.V. Mierz ◽  
R.M. Bayney ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Recombinant Fusion Proteins ◽  
Rapid Characterization

scholarly journals New Opportunities in Glycan Engineering for Therapeutic Proteins

Antibodies ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 5
Author(s):  
Xiaotian Zhong ◽  
Aaron M. D’Antona ◽  
John J. Scarcelli ◽  
Jason C. Rouse
Keyword(s):  
Fusion Proteins ◽  
Therapeutic Proteins ◽  
Quality Attributes ◽  
Efficacy And Safety ◽  
Biological Functions ◽  
Next Generation ◽  
Lysosomal Degradation ◽  
Critical Quality Attributes ◽  
Recombinant Fusion Proteins ◽  
Antibody Diversification

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins.

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