کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020649 1069196 2012 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Improved solubility of replication factor C (RFC) Walker A mutants
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Improved solubility of replication factor C (RFC) Walker A mutants
چکیده انگلیسی

Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.


► Addition of maltose to buffers increases the solubility of RFC complexes.
► Maltose does not affect ATP-dependent PCNA binding, opening, or DNA binding by RFC.
► Maltose disrupts weak ATP-independent interactions between RFC and PCNA.
► PCNA opening, an intramolecular reaction, is two times slower in maltose.
► Maltose prevents protein precipitation and allows for analysis of mutant complexes.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 83, Issue 2, June 2012, Pages 135–144
نویسندگان
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