検索結果 Working with Biotinylated Proteins Studying Protein-Protein Interactions ｜ CellFree Sciences

Working with Biotinylated Proteins

The very strong non-covalent binding of biotin to avidin and streptavidin has been widely used in analytical assays, detection, and protein purification. Adding the biotin ligase BirA from E. coli to wheat germ cell-free protein expression reactions allows for specific mono-biotinylation of proteins having a short recognition sequence for the BirA ligase:

Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.

Matsuoka K. et al..: Proteome Res. 2010 Aug 6;9(8):4264-73.

PMID: 20575507 PMCID: PMC2917173

・Direct preparation of biotinylated proteins by adding BirA to translation reaction

・Development of HTP screening assay on PerkinElmer AlphaScreen™ (Amplified Luminescent Proximity Homogeneous Assay)

・Background free detection without need of protein purification

・Use of biotinylated proteins to screen serum samples for autoimmune antibodies

Specific in situ visualization of plasma cells producing antibodies against Porphyromonas gingivalis in gingival radicular cyst: application of the enzyme-labeled antigen method.

Tsuge S. et al.: J Histochem Cytochem. 2011 Jul;59(7):673-89.

PMID: 21525188 PMCID: PMC3201162

・Biotinylated protein used in different detection assays

・Biotinylated protein used in PerkinElmer AlphaScreen™ experiments (see above)

Profiling of autoantibodies in sera of pancreatic cancer patients.

Nagayoshi Y. et al.: Ann Surg Oncol. 2014 Jun;21 Suppl 3:S459-65.

PMID: 24585405

・Preparation of biotinylated protein library covering 2,183 human genes

・Screening of serum antibodies by PerkinElmer AlphaScreen™ method (see above)

・Identified potential biomarkers for pancreas cancer

Identification of new abscisic acid receptor agonists using a wheat cell-free based drug screening system.

Nemoto K. et al.: Sci Rep. 2018 Mar 9;8(1):4268.

PMID: 29523814 PMCID: PMC5844987

・Abscisic acid (ABA) is main phytohormone involved plant stress response

・Using wheat germ system and PerkinElmer AlphaScreen™ method in a drug screening system for biochemical validation ABA receptors

・Successful use of the system for screening compound library to identify ABA receptor agonists that are candidate agrichemicals

Studying Protein-Protein Interactions

Easy access to modified proteins using a cell-free expression system also provides a great tool to better study protein-protein interactions on different platforms including the PerkinElmer AlphaScreen™ technology or Surface Plasmon Resonance (SPR) that can use biotinylated proteins as mentioned above and for example combines them with tagged proteins to confirm binding of the partners. Protein-protein interactions have been studied with the wheat germ system in plant, animal, and viral research:

Viral Research

Involvement of the 3' Untranslated Region in Encapsidation of the Hepatitis C Virus.

Shi G. et al.: PLoS Pathog. 2016 Feb 11;12(2):e1005441.

PMID: 26867128 PMCID: PMC4750987

・Study on selective packaging of the HCV genome into viral particles

・Identification of 3' UTR element that acts in cis for encapsidation of viral genome

・Using wheat germ system to express FLAG-tagged HCV Core and its mutants to perform RNA binding assays with biotinylated RNA on AlphaScreen™

HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains.

Shibata Y. et al.: PLoS Pathog. 2017 Jan 19;13(1):e1006162.

PMID: 28103322 PMCID: PMC5283754

・Study on Tax protein of human T-cell leukemia virus type 1 (HTLV-1)

・Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex

・Data suggest that Tax could lead to trans-autophosphorylation-mediated IKK activation

・GST, GST-HOIL-1L, GST-HOIP and GST-Sharpin were prepared by wheat germ cell-free protein synthesis during this study

PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation.

Miyakawa K. et al.: Nat Commun. 2019 Apr 23;10(1):1844.

PMID: 31015445 PMCID: PMC6479052

・PIM family of serine/threonine protein kinases phosphorylate Vpx protein

・Using proteomics and functional analysis to show that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions

・Initial screen used a set of 412 human protein kinases

Plant Research

Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues.

Nemoto K. et al.: J Biol Chem. 2015 Jul 3;290(27):16665-77

PMID: 25969537 PMCID: PMC4505418

・Arabidopsis calcium-dependent protein kinase (CDPK/CPK)-related PKs (CRKs) have high Tyr-autophosphorylation activity and can phosphorylate Tyr residue(s) on substrate proteins in Arabidopsis

・Examined autophosphorylation activity of 759 protein kinases using an Arabidopsis protein array and wheat cell-free expression system

・Substrate screening for CRK3 by pulldown assay using protein expressed in wheat cell-free system and protein extracts prepared from cultured Arabidopsis cells

・Found Tyr-autophosphorylation activity for 38 protein kinases included into the screen

Tyrosine phosphorylation of the GARU E3 ubiquitin ligase promotes gibberellin signalling by preventing GID1 degradation.

Nemoto K. et al.: Nat Commun. 2017 Oct 17;8(1):1004.

PMID: 29042542 PMCID: PMC5645313

・Gibberellin (GA) is a major hormone for plant growth and development

・Study shows that GARU (GA receptor RING E3 ubiquitin ligase) mediates ubiquitin-dependent degradation of GID1

・TAGK2 plant Tyr-kinase is a target of genistein and inhibits GARU-GID1A interactions by phosphorylation of GARU at Tyr321

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions

OsMYC2, an essential factor for JA-inductive sakuranetin production in rice, interacts with MYC2-like proteins that enhance its transactivation ability.

Ogawa S. et al.: Sci Rep. 2017 Jan 9;7:40175.

PMID: 28067270 PMCID: PMC5220304

・Jasmonic acid (JA) signaling induces basic helix-loop-helix transcriptional factor OsMYC2 and enhanced the activity of the OsNOMT promoter

・OsMYC2 interacts with OsMYL1 and OsMYL2 to further enhance transactivation activity of OsMYC2

・OsMYL1 and OsMYL2 support JA signaling via OsMYC2 and play role in production of sakuranetin in rice

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions

Animal Research

Bach2-Batf interactions control Th2-type immune response by regulating the IL-4 amplification loop.

Kuwahara M. et al.: Nat Commun. 2016 Sep 1;7:12596.

PMID: 27581382 PMCID: PMC5025763

・Study shows that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions