Heterologous high-level E. coli expression, purification and biophysical characterization of the spine-associated RapGAP (SPAR) PDZ domain

Brown, Breann L.; Hadley, Michael S.; Page, Rebecca

doi:10.1016/j.pep.2008.07.003

Cited by 11 publications

(11 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, decades of work by individual protein chemists, coupled with the recent experiences of high‐throughput structural genomics efforts, have resulted in the identification of a consensus protocol that allows a diverse set of proteins to be successfully expressed in E. coli . A flowchart of the protocol typically used by the authors to express a diverse set of proteins is shown in Figure (Mustelin et al, ; Brown et al, ; Critton et al, ). More extensive protocols are also available (Peti and Page, ; Gråslund et al, ).…”

Section: Representative Protocol For Expressing Proteins In Bacteriamentioning

confidence: 99%

Strategies to Optimize Protein Expression in E. coli

2010

Self Cite

View full text Add to dashboard Cite

Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post-translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli.

show abstract

Section: Representative Protocol For Expressing Proteins In Bacteriamentioning

confidence: 99%

Strategies to Optimize Protein Expression in E. coli

2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…The GST is 211 amino acids (roughly 28 kDa) in size. Glutathione Stransferase tags are generally used for expression and purification and which is not used as a solubility enhancing tag (Esposito and Chatterjee, 2006;Brown et al 2008). GST tags are widely used due to their specific and robust binding towards glutathione agarose and allow single-step purification of expressed protein (Smith and Johnson, 1988).…”

Section: A Glutathione S-transferase (Gst)mentioning

confidence: 99%

Effect of supplementing Moringa oleifera essential oils on milk quality and fatty acid profile in dairy sheep

Hemamalini

Ezhilmathi

Mercy

2019

IJAR

View full text Add to dashboard Cite

Escherichia coli is the most extensively used organism in recombinant protein production. It has several advantages including a very short life cycle, ease of genetic manipulation and the well-known cell biology etc. which makes E. coli as the perfect host for recombinant protein expression. Despite many advantages, E. coli also have few disadvantages such as coupled transcription and translation and lack of eukaryotic post-translational modifications. These challenges can be overcome by adopting several strategies such as, using different E. coli expression vectors, changing the gene sequence without altering the functional domain, modified E. coli strain usage, changing the culture parameters and co-expression with a molecular chaperone. In this review, we present the level of strategies used to enhance the recombinant protein expression and its stability in E. coli.

show abstract

“…Many scaffold proteins, including Spa-1 and SPAR contain at least one domain in common, the PDZ domain, which binds the carboxyl terminus of a variety of proteins, including those of transmembrane receptors and other PDZ domains [91]. The Rap-effector AF-6 can simultaneously bind RapGTP and Rap1GAP or Spa-1 to recruit GAP activity to Rap within the same molecular complex, the binding of AF-6 with Spa-1 takes place at PDZ domain, which is also contained by AF-6 [92].…”

Section: Spa-1 and Sparmentioning

confidence: 99%

Rap-Interacting Proteins are Key Players in the Rap Symphony Orchestra

Guo¹,

An²,

Yang³

et al. 2016

Cell Physiol Biochem

View full text Add to dashboard Cite

Rap, a member of the Ras-like small G-protein family, is a key node among G-protein coupled receptors (GPCR), receptor tyrosine kinases (RTKs), ion channels and many other downstream pathways. Rap plays a unique role in cell morphogenesis, adhesion, migration, exocytosis, proliferation, apoptosis and carcinogenesis. The complexity and diversity of Rap functions are tightly regulated by Rap-interacting proteins such as GEFs, GAPs, Rap effectors and scaffold proteins. These interacting proteins decide the subcellular localization of Rap, the interaction modes with downstream Rap effectors and tune Rap as an atypical molecular conductor, coupling extra- and intracellular signals to various pathways. In this review, we summarize four groups of Rap-interacting proteins, highlight their distinctions in Rap-binding properties and interactive modes and discuss their contribution to the spatiotemporal regulation of Rap as well as the implications of targeting Rap-interacting proteins in human cancer therapy.

show abstract

Heterologous high-level E. coli expression, purification and biophysical characterization of the spine-associated RapGAP (SPAR) PDZ domain

Cited by 11 publications

References 20 publications

Strategies to Optimize Protein Expression in E. coli

Strategies to Optimize Protein Expression in E. coli

Effect of supplementing Moringa oleifera essential oils on milk quality and fatty acid profile in dairy sheep

Rap-Interacting Proteins are Key Players in the Rap Symphony Orchestra

Contact Info

Product

Resources

About