Different effects of L-arginine on protein refolding: suppressing aggregates of hydrophobic interaction, not covalent binding.
Arginine is one of the most favorable additives in protein refolding. However, arginine does not work for certain disulfide-bond-containing proteins, which is not yet well explained. In this work, refolding of three proteins in the presence of 0-2 M arginine was investigated and compared. Bovine carbonic anhydrase B (CAB), containing no cysteine, was successfully refolded with the help of arginine. The refolding yield could reach almost 100% in the presence of 0.75 M arginine. However, recombinant human colony stimulating factor (rhG-CSF), containing five cysteines, could only achieve 65% refolding yield. The formation of aggregates was found. Blocking of free SH groups of the denatured rhG-CSF by iodoacetamide and subsequently refolding of the protein could reduce the aggregate formation substantially. Further investigation on recombinant green fluorescence protein (GFP), containing two cysteines, also revealed the accumulation of oligomers. The content of oligomers increased with the concentration of arginine, reaching about 30% at 2 M arginine. Comparison of reduced and nonreduced SDS-PAGE revealed that the oligomers were formed through intermolecular disulfide binding. Analysis of the refolding kinetics indicated that intermolecular disulfide bonds were probably formed in the intermediate stage where arginine slowed down the refolding rate and stabilized the intermediates. The accumulated intermediates with unpaired cysteine possessed more chances to react with each other to form oligomers, whereas arginine failed to inhibit disulfide bond formation.
Chen
Jing
J
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China.
Liu
Yongdong
Y
Wang
Yinjue
Y
Ding
Hong
H
Su
Zhiguo
Z
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D024401:Carbonic Anhydrase I / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000187:drug effects* / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000187:drug effects*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D017510:Protein Folding / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D049452:Green Fluorescent Proteins; D001120:Arginine; D024401:Carbonic Anhydrase I