About: High quality protein microarray using in situ protein purification

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An Entity of Type : schema:ScholarlyArticle, within Data Space : covidontheweb.inria.fr associated with source document(s)

Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
has title	High quality protein microarray using in situ protein purification
Creator	Fleischmann, Robert Pandya, Gagan Peterson, Scott Grose, Carissa Kwon, Keehwan Pieper, Rembert
Source	Medline; PMC
abstract	BACKGROUND: In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. RESULTS: Two slide surfaces were examined, chelated Cu(2+ )slides suspended on a polyethylene glycol (PEG) coating and chelated Ni(2+ )slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu(2+ )slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays. CONCLUSION: An optimized platform for in situ protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.
has issue date	2009-08-23(xsd:dateTime)
bibo:doi	10.1186/1472-6750-9-72
bibo:pmid	19698181
has license	cc-by
sha1sum (hex)	2a8339ac2c0891d5113f221ebd214f2b1d903d38
schema:url	https://doi.org/10.1186/1472-6750-9-72
resource representing a document's title	High quality protein microarray using in situ protein purification
has PubMed Central identifier	PMC2746808
has PubMed identifier	19698181
schema:publication	BMC Biotechnol
resource representing a document's body	covid:2a8339ac2c0891d5113f221ebd214f2b1d903d38#body_text
is schema:about of	named entity 'IMAC' named entity 'microarrays' named entity 'surfaces' named entity 'protein microarray' covid:arg/2a8339ac2c0891d5113f221ebd214f2b1d903d38 named entity 'tags' named entity 'format' named entity 'slides' named entity 'metal' named entity 'screening' named entity 'process' named entity 'protein microarrays' named entity 'gold standard' named entity 'protein expression' named entity 'protein' named entity 'protein microarray' named entity 'protein purification' named entity 'S. pneumoniae' named entity 'iron' named entity 'antibodies' named entity 'fusion proteins' named entity 'metal chelate' named entity 'membrane proteins' named entity 'antiserum' named entity 'Saco' named entity 'His-tagged' named entity 'His-tagged' named entity 'competitive binding' named entity 'microarray chip' named entity 'ORFs' named entity 'LacI' named entity 'High affinity' named entity 'quantitative approach' named entity 'LacI' named entity 'antibody' named entity 'IPTG' named entity 'recombinant protein' named entity 'E. coli' named entity 'transmembrane domain' named entity 'expression vector' named entity 'gold standard' named entity 'FolE' named entity 'lysis' named entity 'C-terminal' named entity 'FolE' named entity 'antibodies' named entity 'LacI' named entity 'recombinant proteins' named entity 'PBS buffer' named entity 'protein microarray' named entity '0.9' named entity 'non-specific' named entity 'His-tag' named entity 'PBS' named entity 'protein microarray' named entity 'chelate' named entity 'antibody' named entity 'immunogenic' named entity 'Alexa Fluor' named entity 'protein expression' named entity 'protein microarray' named entity 'S. pneumoniae' named entity 'secondary antibody' named entity 'endogenous proteins' named entity 'FolE' named entity 'protein purification' named entity 'E. coli' named entity 'surface chemistry' named entity 'endogenous proteins' named entity 'Lac repressor' named entity 'Alexa Fluor' named entity 'polyethylene glycol' named entity 'cis' named entity 'microbial' named entity 'S. pneumoniae' named entity 'microtiter plates'

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