Expression and Purification of Homogenous Proteins in Saccharomyces cerevisiae Based on Ubiquitin-FLAG Fusion

Einhauer, A.; Schuster, Martin; Wasserbauer, Erich; Jungbauer, Alois

doi:10.1006/prep.2001.1595

Cited by 38 publications

(15 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A unique aspect of FLAG utilization is the inherent enterokinase cleavage site located within the five C-terminal residues of the peptide sequence [91], which is ad-vantageous if the recombinant protein of interest is intended for therapeutic use, since the epitope itself is immunogenic. The FLAG peptide sequence is recognized by the antibody M1, in either a calcium-dependent [92], or independent [93], manner; however, additional commercial mAb choices include M2 and M5, which comprise slightly different recognition sites and affinity. FLAG-tagged fusion proteins can be purified using an immobilized monoclonal antibody matrix under non-denaturing conditions and eluted by lowering the pH or adding chelating agents such as EDTA.…”

Section: C-myc Ha and Flagmentioning

confidence: 99%

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Young

Britton

Robinson

2012

Biotechnology Journal

410

267

View full text Add to dashboard Cite

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.

show abstract

Section: C-myc Ha and Flagmentioning

confidence: 99%

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Young

Britton

Robinson

2012

Biotechnology Journal

410

267

View full text Add to dashboard Cite

show abstract

“…Yeast cells were lysed by glass beads method [23]. The protein content in the supernatants was estimated by dye binding method [24], using bovine serum albumin as the standard protein.…”

Section: Yeast Cell Lysismentioning

confidence: 99%

Gpd1 Regulates the Activity of Tcp-1 and Heat Shock Response in Yeast Cells: Effect on Aggregation of Mutant Huntingtin

Bhadra

Roy

2015

Mol Neurobiol

View full text Add to dashboard Cite

A significant correlation has been observed between the length of the polyglutamine tract in huntingtin, its aggregation and the progression of Huntington's disease (HD). The chaperonin TRiC is a potent antagonist of aggregation of mutant huntingtin. Using the well-validated Saccharomyces cerevisiae model of HD, we have investigated the role of age-related post-translational modifications of this heterooligomeric chaperonin on its ability to inhibit aggregation of the mutant protein. We show that the glycerol synthetic enzyme Gpd1 is involved in the post-translational modification of Tcp-1 (subunit of TRiC) by acetylation and glycation through the NAD(+)/NADH shuttle and the triose phosphate intermediate dihydroxyacetone phosphate, respectively. The extent of modification of Tcp-1 shows a negative correlation with the solubility of mutant huntingtin. The absence of Gpd1 also induces heat shock response in yeast cells, further inhibiting aggregation of the mutant protein. Thus, Gpd1 acts as a major regulator of the protein folding machinery in the yeast model of HD. Modification and inactivation of cellular chaperonin are accelerated in an aging cell, which has further deleterious effects for a cell harbouring misfolded/aggregated protein(s).

show abstract

“…Cell pellets were lysed using acid-treated glass beads (Einhauer et al 2002). Lysates were set aside for 1 h for the glass beads and debris to settle down.…”

Section: Native Page Analysismentioning

confidence: 99%

Activation of salt shock response leads to solubilisation of mutant huntingtin in Saccharomyces cerevisiae

Saleh

Bhadra

Roy

2014

Cell Stress and Chaperones

View full text Add to dashboard Cite

Formation of cytoplasmic and nuclear aggregates is a hallmark of Huntington's disease (HD). Inhibition of aggregation of mutant huntingtin has been suggested to be a feasible approach to slow down the progress of this neurodegenerative disorder. Exposure to environmental stimuli leads to the activation of the stress response machinery of the cell. In this work, we have investigated the effect of salt shock on the aggregation of mutant huntingtin (103Q-htt) in a yeast model of HD. We found that at an optimum concentration of NaCl, the protein no longer formed aggregates and existed in the soluble form. This led to lower oxidative stress in the cell. Salt shock resulted in the synthesis of the osmolyte glycerol, which was partially responsible for the beneficial effect of stress. Surprisingly, we also found increase in the synthesis of another osmolyte, trehalose. Using deletion strains, we were able to show that the effect on solubilisation of mutant huntingtin is due to the synthesis of optimum amounts of both osmolytes. Stress-induced effect was monitored on gene expression. Genes related to proteins of the osmosensory pathway were upregulated on exposure to salt while those coding for stress response proteins were downregulated when solubilisation of mutant huntingtin occurred. Our study shows that activation of stress response elements can have beneficial effect in the solubilisation of huntingtin in a yeast model of HD.

show abstract

Expression and Purification of Homogenous Proteins in Saccharomyces cerevisiae Based on Ubiquitin-FLAG Fusion

Cited by 38 publications

References 27 publications

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Gpd1 Regulates the Activity of Tcp-1 and Heat Shock Response in Yeast Cells: Effect on Aggregation of Mutant Huntingtin

Activation of salt shock response leads to solubilisation of mutant huntingtin in Saccharomyces cerevisiae

Contact Info

Product

Resources

About