Control of aggregation in protein refolding: cooperative effects of artificial chaperone and cold temperature.
Refolding of GuHCl-denatured recombinant-human growth hormone (r-hGH) was investigated in both dilution additive and artificial chaperone assisted modes. In both techniques, it was found that CTAB is a better additive (in dilution mode) or a capturing agent (in artificial chaperone method). Neither of the two techniques was capable of complete inhibition of aggregates formed during refolding process. In dilution, using CTAB or alpha-cyclodextrin (alpha-CD) as two different additives, the aggregation was inhibited by almost 55%. However, the extent of inhibition raised to almost 82% in artificial chaperone assisted mode using CTAB as the capturing and alpha-CD as the stripping agents. Maximum inhibition of aggregation (up to 97%) was obtained when the entire process of refolding was done at 4 degrees C. However, under this temperature program, the far-UV CD and intrinsic fluorescence spectra of the refolded samples were not superimposable on their respective native spectra. The spectra superimposibilities were obtained when the refolding process was achieved under a well worked out temperature program: incubation of the sample for 3 min at 4 degrees C after initiation of the stripping step followed by overnight incubation at 22 degrees C. Based on these data, it is expected that higher activity recovery yields of recombinant proteins, particularly at relatively higher protein concentrations, could be achieved by getting a better molecular understanding of major factors responsive for aggregation and refolding pathways.
Yazdanparast
Razieh
R
Institute of Biochemistry and Biophysics, The University of Tehran, Tehran, Iran. yazdan@ibb.ut.ac.ir
Esmaeili
Mohammad Ali
MA
Khodagholi
Fariba
F
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology*; D003080:Cold Temperature; D013006:Growth Hormone / Q000737:chemistry* / Q000187:drug effects*; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D047391:alpha-Cyclodextrins / Q000494:pharmacology
D002593:Cetrimonium Compounds; D047391:alpha-Cyclodextrins; D013006:Growth Hormone; D019791:Guanidine; C032613:alpha-cyclodextrin; D000077286:Cetrimonium