A novel "reverse screening" to identify refolding additives for activin-A.
A general approach for refolding recombinant proteins from inclusion bodies (IBs) is to screen conditions, that facilitate a conversion of unfolded to folded structure and minimize a conversion of unfolded to misfolded and aggregated structures. In this simplified model, such conditions may be those that stabilize the native protein and/or reduce aggregation. In this paper, a novel screening approach, termed reverse screening, was developed using a native activin. Activin-A, a member of transforming growth factor beta superfamily, is a homodimeric protein with nine disulfide bonds. We examined partial unfolding process of native activin-A dissolved in a buffer containing moderate concentrations of denaturant and reducing reagent (i.e., 1.5 M urea and 0.2 mM dithiothreitol). The recovery of the protein was followed by reverse-phase high performance chromatography analysis. Without additives, activin-A showed about 60% loss of the protein due to aggregation after 12-h incubation in the above condition. We then tested various additives for their effects on the recovery after partial unfolding. One of these additives, sodium taurodeoxycholate (TDCA), greatly increased recovery and suppressed aggregation of the protein. These additives were then tested for refolding activin-A from IBs. TDCA among others is proved to be a highly effective refolding additive. These results strongly suggest that reverse screening using native proteins, if available, may be another approach to discovering effective refolding additives.
Ejima
Daisuke
D
Applied Research Department, Amino Science Laboratories, Ajinomoto Co., Inc., The University of Tokyo, Kawasaki 210-8681, Japan. daisuke_ejima@ajinomoto.com
Ono
Kunio
K
Tsumoto
Kouhei
K
Arakawa
Tsutomu
T
Eto
Yuzuru
Y
D028341:Activins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D002479:Inclusion Bodies / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D013657:Taurodeoxycholic Acid / Q000494:pharmacology
D007202:Indicators and Reagents; C422405:activin A; D028341:Activins; D013657:Taurodeoxycholic Acid