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

, : Improved solubility of replication factor C (RFC) Walker A mutants. Protein Expression and Purification 83(2): 135-144

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.


PMID: 22469630

DOI: 10.1016/j.pep.2012.03.010

Other references

Sivasithamparam, K.; Parker, C.A., 1976: Production of chlamydo spores and micro sclerotia by gaeumannomyces graminis var tritici. The purpose of the study was to see if this pathogen was able to produce microscopic resting structures which could survive in soil. Colonies were allowed to grow for 5 days on potato dextrose agar and then subjected to pH and osmotic shock treatm...

Ratnavel, R.C.; Mclean, W.H.I.; Cerio, R.; Griffiths, W.A.D.; Leigh, I.M., 1996: The molecular pathology of Jackson-Lawler pachyonychia congenita. Journal of Cutaneous Pathology 23(1): 91

Fales, H.M.; Milne, G.W.; Pisano, J.J.; Brewer, H.B.; Blum, M.S.; MacConnell, J.G.; Brand, J.; Law, N., 1972: Biological applications of electron ionization and chemical ionization mass spectrometry. Recent Progress in Hormone Research 28: 591-626

Rice, J., 2008: All the sourcing not fit to print: citing electronic material in your article. Chest 133(6): 1524-1526

Elhawary, T.M.; El-Bendary, A.S.; Demerdash, H., 2012: Maternal serum endoglin as an early marker of pre-eclampsia in high-risk patients. Pre-eclampsia is a potentially serious condition that still accounts for significant morbidity and mortality for the affected mother and neonate. Although the pathogenesis is not fully understood, it is now widely accepted that vascular endothelia...

Vartiainen, T.; Liimatainen, A., 1986: High levels of mutagenic activity in chlorinated drinking water in Finland. The organic extract from the drinking water of Kuopio, Eastern Finland, turned out to be highly mutagenic in the Ames test. The direct mutagenic activities of the acid/neutral fractions of 48 drinking water samples were on an average 1700 net reve...

Stermitz, F.R.; Lorenz, P.; Tawara, J.N.; Zenewicz, L.A.; Lewis, K., 2000: Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor. Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants,...

Kamita, G.; Kolle, M.; Huang, F.; Baumberg, J.J.; Steiner, U., 2012: Multilayer mirrored bubbles with spatially-chirped and elastically-tuneable optical bandgaps. We demonstrate the multifolding Origami manufacture of elastically-deformable Distributed Bragg Reflector (DBR) membranes that reversibly color-tune across the full visible spectrum without compromising their peak reflectance. Multilayer films com...

Senturia, B.H., 1950: Diffuse external otitis; its pathology and treatment. Transactions - American Academy of Ophthalmology and Otolaryngology. American Academy of Ophthalmology and Otolaryngology 55: 147-159

Wecker, L.; Laskowski, B.; Dettbarn, W.D., 1978: Neuromuscular dysfunction induced by acetylcholinesterase inhibition. The organophosphate cholinesterase inhibitor paraoxon produces a dose-dependent necrosis in rat skeletal muscle fibers after a single administration. The pathology, which is initiated at the motor end-plate region, is evident as early as 30 minute...