In vitro-refolding of a single-chain Fv fragment in the presence of heteroaromatic thiols.
The aim of the present work was to explore the use of heteroaromatic thiol compounds, namely derivatives of pyridine and pyrimidine, as redox reagents for the in vitro-refolding of a recombinantly expressed single-chain Fv fragment (scFvOx). The mixed disulfide of scFvOx with glutathione was used as a starting material, while reduced glutathione, 4-mercaptopyridine, 2-mercaptopyrimidine, 2-mercaptopyridine N-oxide, and the mercaptobenzene derivative thiosalicylic acid, respectively, served as catalysts for the formation of native disulfide bonds during renaturation. In contrast to thiosalicylic acid, and despite their significantly lower thiol pKa values, none of the heteroaromatic thiol compounds accelerated the apparent kinetics of in vitro-refolding compared to the naturally occurring peptide glutathione. However, significantly improved renaturation yields were observed in the presence of 4-mercaptopyridine and 2-mercaptopyrimidine, demonstrating the usefulness of aromatic thiol compounds as reagents for the in vitro-refolding of antibody fragments.
Patil
Ganesh
G
Institut für Biotechnologie, Martin-Luther-Universität Halle/Wittenberg, Kurt-Mothes-Str.3, 06120 Halle (Saale), Germany.
Rudolph
Rainer
R
Lange
Christian
C
D002021:Buffers; D002384:Catalysis; D004220:Disulfides / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D010084:Oxidation-Reduction; D017510:Protein Folding; D020673:Protein Renaturation; D011725:Pyridines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D012459:Salicylates / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013871:Thiones / Q000737:chemistry
D002021:Buffers; D004220:Disulfides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011725:Pyridines; D011743:Pyrimidines; D012459:Salicylates; D013438:Sulfhydryl Compounds; D013871:Thiones; C064001:pyrimidine-2-thiol; C042861:4-thiopyridine; C008704:pyrithione; C441201:thiosalicylic acid; D005978:Glutathione