7saj: Difference between revisions
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==Crystal Structure of LaM2 Nanobody bound to mCherry== | |||
<StructureSection load='7saj' size='340' side='right'caption='[[7saj]], [[Resolution|resolution]] 2.37Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[7saj]] is a 10 chain structure with sequence from [https://en.wikipedia.org/wiki/Discosoma_sp. Discosoma sp.] and [https://en.wikipedia.org/wiki/Lama_glama Lama glama]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7SAJ OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7SAJ FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=NRQ:{(4Z)-4-(4-HYDROXYBENZYLIDENE)-2-[3-(METHYLTHIO)PROPANIMIDOYL]-5-OXO-4,5-DIHYDRO-1H-IMIDAZOL-1-YL}ACETIC+ACID'>NRQ</scene></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=7saj FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7saj OCA], [https://pdbe.org/7saj PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7saj RCSB], [https://www.ebi.ac.uk/pdbsum/7saj PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7saj ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[[https://www.uniprot.org/uniprot/A0A366VY15_9GAMM A0A366VY15_9GAMM]] | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2alpha expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed. | |||
High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices.,Cong ATQ, Witter TL, Schellenberg MJ Protein Sci. 2022 Sep;31(9):e4383. doi: 10.1002/pro.4383. PMID:36040252<ref>PMID:36040252</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: Cong | <div class="pdbe-citations 7saj" style="background-color:#fffaf0;"></div> | ||
[[Category: Schellenberg | == References == | ||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Discosoma sp]] | |||
[[Category: Lama glama]] | |||
[[Category: Large Structures]] | |||
[[Category: Cong ATQ]] | |||
[[Category: Schellenberg MJ]] | |||