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Improved solubility of replication factor C (RFC) Walker A mutants.

Melissa R Marzahn¹, Linda B Bloom

¹University of Florida, Department of Biochemistry & Molecular Biology, 1600 SW Archer Road, P.O. Box 100245, Gainesville, FL 32610-0245, USA.

Protein Expression and Purification

|April 4, 2012

Summary

Adding maltose to buffers significantly enhances the solubility of replication factor C (RFC) and its mutants. This finding is crucial for studying RFC

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Area of Science:

Biochemistry
Molecular Biology
Protein Chemistry

Background:

Protein insolubility is a major challenge in recombinant protein expression and purification, particularly for eukaryotic proteins in bacterial systems.
The replication factor C (RFC) complex from Saccharomyces cerevisiae is known to have limited solubility, further exacerbated in certain mutant forms.
Mutant RFC forms with point mutations in the Walker A motif exhibit even lower solubility than the wild-type complex.

Purpose of the Study:

To investigate methods for improving the solubility of replication factor C (RFC) and its mutants for biochemical analysis.
To assess the impact of maltose as a buffer additive on RFC solubility and enzyme activity.
To validate the use of maltose in fluorescence-based assays for studying RFC function.

Main Methods:

Expression and purification of wild-type and mutant Saccharomyces cerevisiae replication factor C (RFC) complexes.
Solubility assays with and without the addition of 0.75 M maltose in storage and assay buffers.
Characterization of RFC clamp loading activity using environmentally sensitive fluorescence-based assays.
Control experiments to evaluate the effect of maltose on fluorophore response and enzyme kinetics.

Main Results:

The addition of 0.75 M maltose to storage and assay buffers substantially increased the solubility of both wild-type and mutant RFC.
Maltose prevented the dissociation of the RFC complex, maintaining its structural integrity.
Maltose did not interfere with the fluorescence signals in the assays or alter the enzymatic activity of wild-type RFC.

Conclusions:

Maltose is an effective and compatible additive for enhancing the solubility and stability of replication factor C (RFC) and its mutants.
The use of maltose enables robust downstream analysis, including fluorescence-based enzyme assays, of challenging RFC mutants.
This strategy facilitates the biochemical characterization of RFC function and the study of mutations affecting its activity.