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Reversible formation of on-pathway macroscopic aggregates during the folding of maltose binding protein

C Ganesh1, F N Zaidi, J B Udgaonkar

  • 1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.

Protein Science : a Publication of the Protein Society
|July 27, 2001
PubMed

Insights

During maltose binding protein (MBP) refolding, transient aggregates form and spontaneously disaggregate. These aggregates yield active protein, contributing to overall refolding efficiency.

Area of Science:

  • Biochemistry
  • Protein Folding
  • Molecular Biology

Background:

  • Maltose binding protein (MBP) is a crucial model for studying protein folding and export mechanisms.
  • Understanding protein aggregation during refolding is vital for protein therapeutics and biotechnology.

Purpose of the Study:

  • To investigate the transient formation and spontaneous disaggregation of macroscopic aggregates during maltose binding protein (MBP) refolding.
  • To characterize the nature of these aggregates and their contribution to the final yield of active protein.

Main Methods:

  • Micromolar concentrations of MBP were refolded, and aggregate formation was monitored.
  • Centrifugation was used to separate soluble and aggregated protein phases.
  • Spectroscopic probes (optical and electron microscopy) were employed to analyze aggregate structure and kinetics.
  • The effect of chaperones like SecB and GroEL on aggregation was assessed.

Main Results:

  • Macroscopic aggregates form transiently during MBP refolding at micromolar concentrations.
  • These aggregates spontaneously disaggregate, yielding refolded protein with native structure and activity.
  • Both soluble and aggregated phases contribute to the total refolded protein yield.
  • SecB chaperone reduced but did not eliminate aggregation; GroEL had no effect.
  • Aggregation proceeds via a collapsed intermediate with secondary structure; aggregates can convert directly to a native state.
  • MBP aggregates are not amyloid structures.

Conclusions:

  • Transient aggregation during MBP refolding is a reversible process that contributes to the overall yield of active protein.
  • Chaperone interactions, specifically with SecB, can modulate but not abolish this aggregation pathway.
  • The findings provide insights into protein folding dynamics and strategies for optimizing recombinant protein production.

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