Comparison of the oxidative folding of lysozyme at a high protein concentration using aromatic thiols versus glutathione.
The production of proteins using recombinant DNA technology often requires the use of in vitro protein folding. In order to facilitate in vitro protein folding, a redox buffer is added to the protein folding mixture. The redox buffer is composed of a small molecule disulfide and/or a small molecule thiol. Recently, redox buffers containing aromatic thiols have been shown to be an improvement over traditional redox buffers such as glutathione. For in vitro protein folding to be relevant to protein production on a larger scale, high protein concentrations are required to avoid large volumes of folding buffer. Therefore, we investigated the in vitro folding of lysozyme at 1 mg/mL instead of the traditional 0.1 mg/mL. Aromatic thiols and aromatic disulfides were compared directly with glutathione and glutathione disulfide, the most commonly used redox buffer. Folding experiments at pH 7 using aromatic thiols increased the yield by 20-40% and the folding rate constants by as much as 11 times relative to glutathione. At pH 8, improvements in yields of up to 25% and up to a 7-fold increase in folding rate constants were demonstrated. The effect of aromatic disulfide concentration was also investigated.
Madar
David J
DJ
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.
Patel
Amar S
AS
Lees
Watson J
WJ
D000818:Animals; D002645:Chickens; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D009113:Muramidase; D005978:Glutathione; D019791:Guanidine