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Question about yield of refolded proteins

Posted by waughd on 11 Mar 2013 at 18:07 GMT

Hi. I would be very interested to know if the yield of properly folded MSP1 obtained by refolding of the untagged vs. the MBP-tagged protein was any different on a mole per mole basis (moles recovered of properly folded vs. moles added to the refolding experiment). Thanks, Dave Waugh.

No competing interests declared.

RE: Question about yield of refolded proteins

chaffott replied to waughd on 22 Mar 2013 at 10:43 GMT

Dear Dr Waugh,

Thank you for your interesting question which raises an important detail that we should have included in our article. As mentioned in the text, the yield (molar basis) of properly folded F19 within the MBP fusion was at least 70 %. This percentage is a lower limit that takes into account the high homogeneity (above 95 %) as deduced from the NMR spectra and the processing of the fusion protein to recover the F19 moiety (proteolytic cleavage, purification steps, dialysis, etc.). Moreover, because of the high concentrations needed for NMR, a small amount of aggregated, correctly and incorrectly folded protein (also observed with native F19 obtained from periplasmic or insect cell expression) had been removed by centrifugation. In the case of the isolated F19 fragment, similarly taking into consideration the conditioning of the sample before NMR experiments (which in this case was limited to dialysis, freeze drying and solubilisation at high concentration), the global yield of recovery was at least 85 %. In both cases (with or without MBP), the real refolding yield was probably higher than 85 %. It is interesting to note that the assistance of MBP to the in vivo folding of F19 that we evidenced upon periplasmic expression (Ref.) is not detected in the in vitro refolding context. This discrepancy may be related to the difference between the in vivo and in vitro experimental conditions (coupling between translocation) and folding, redox machinery, etc.). In vivo, the refolding/oxidation reaction may more likely be kinetically rather than thermodynamically controlled.

No competing interests declared.

RE: RE: Question about yield of refolded proteins

chaffott replied to chaffott on 22 Mar 2013 at 11:04 GMT

Dear Dr Waugh,

I apologize having forgotten to insert the reference in parentheses in my posted response to your question: it is the reference 16 in the article (Planson et al (2003) Biochemistry 42: 13202–13211). Sorry...

A. C.

No competing interests declared.