Modification of an expression vector for efficient recombinant production and purification of mitogillin of Aspergillus fumigatus expressed in Escherichia coli

Al-Sheikh, Ali; Ahmad, Suhail; Khan, Zia U.

doi:10.1016/j.pep.2010.09.014

Cited by 1 publication

(1 citation statement)

References 39 publications

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“…The commonly used pGEX‐6P GST expression vectors (#28–9546–48, 50, 51; GE Healthcare, Sydney, Australia) feature a gene encoding GST from the parasite Schistosoma japonicum and a cleavage site for the human rhinovirus 3C protease , marketed by GE Healthcare as PreScission protease. These vectors and the PreScission protease have been used extensively for recombinant protein production and purification .…”

Section: Representative Ms Analysis Of Gst‐rab31 Pull‐downs By Boilinmentioning

confidence: 99%

Single‐step protease cleavage elution for identification of protein–protein interactions from GST pull‐down and mass spectrometry

et al. 2014

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The study of protein-protein interactions is a major theme in biological disciplines. Pull-down or affinity-precipitation assays using GST fusion proteins have become one of the most common and valuable approaches to identify novel binding partners for proteins of interest (bait). Non-specific binding of prey proteins to the beads or to GST itself, however, inevitably complicates and impedes subsequent analysis of pull-down results. A variety of measures, each with inherent advantages and limitations, can minimise the extent of the background. This technical brief details and tests a modification of established GST pull-down protocols. By specifically eluting only the bait (minus the GST tag) and the associated non-specific binding proteins with a simple, single-step protease cleavage, a cleaner platform for downstream protein identification with MS is established. We present a proof of concept for this method, as evidenced by a GST pull-down/MS case study of the small guanosine triphosphatase (GTPase) Rab31 in which: (i) sensitivity was enhanced, (ii) a reduced level of background was observed, (iii) distinguishability of non-specific contaminant proteins from genuine binders was improved and (iv) a putative new protein-protein interaction was discovered. Our protease cleavage step is readily applicable to all further affinity tag pull-downs.

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Section: Representative Ms Analysis Of Gst‐rab31 Pull‐downs By Boilinmentioning

confidence: 99%

Single‐step protease cleavage elution for identification of protein–protein interactions from GST pull‐down and mass spectrometry

et al. 2014

View full text Add to dashboard Cite

show abstract

Modification of an expression vector for efficient recombinant production and purification of mitogillin of Aspergillus fumigatus expressed in Escherichia coli

Cited by 1 publication

References 39 publications

Single‐step protease cleavage elution for identification of protein–protein interactions from GST pull‐down and mass spectrometry

Single‐step protease cleavage elution for identification of protein–protein interactions from GST pull‐down and mass spectrometry

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