Recently I was thinking about my RNA project and wishing that I could express RNA in bacteria as is typically done for recombinant proteins. I looked at the July issue of Nature Methods to see a paper describing exactly the technology I was imagining. The authors created plasmids which allow the fusion of a target RNA sequence to a tRNA scaffold which is authenically processed by the cellular machinery and protected from degradation by the intrinsic stability of the tRNA. The potential benefits of this technology for NMR of RNA are:
1. low cost expression of uniformly-labeled RNA
2. the tRNA scaffold acts to stabilize the target RNA
3. the RNA structure may be preserved during purification
4. affinity purification of RNA
Their is also an informative “news and views” piece discussing the work presented. Looking back over their previous work it is clear that the authors have worked on this technology for quite some time. Publication in this highly visible forum will hopefully give the technique exposure which will encourage further developments.
As for me, I am already ordering primers to use this new technology! Please post to the forum if you have any comments or questions.
Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
Protein Expr Purif. 2011 May 13;
Authors: Bellot G, Pascal R, Mendre C, Urbach S, Mouillac B, Déméné H
The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules,...
[NMR paper] Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloi
Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR.
Related Articles Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR.
Protein Expr Purif. 2005 Jul;42(1):200-10
Authors: Sharpe S, Yau WM, Tycko R
Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram...
nmrlearner
Journal club
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11-24-2010 11:14 PM
[NMR paper] Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris
Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR.
Related Articles Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR.
Protein Expr Purif. 2004 Oct;37(2):336-43
Authors: Barral P, Tejera ML, Treviño MA, Batanero E, Villalba M, Bruix M, Rodríguez R
Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen...
nmrlearner
Journal club
0
11-24-2010 10:01 PM
[NMR paper] Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glyco
Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.
Related Articles Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.
Protein Expr Purif. 2000 Aug;19(3):335-42
Authors: Cubeddu L, Moss CX, Swarbrick JD, Gooley AA, Williams KL, Curmi PM, Slade MB, Mabbutt BC
The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically...
nmrlearner
Journal club
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11-19-2010 08:29 PM
[NMR paper] Recombinant gene expression and 1H NMR characteristics of the kringle (2 + 3) supermo
Recombinant gene expression and 1H NMR characteristics of the kringle (2 + 3) supermodule: spectroscopic/functional individuality of plasminogen kringle domains.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Recombinant gene expression and 1H NMR characteristics of the kringle (2 + 3) supermodule: spectroscopic/functional individuality of plasminogen kringle domains.
Biochemistry. 1996 Feb 20;35(7):2357-64
Authors: Söhndel S, Hu CK, Marti D, Affolter M, Schaller J, Llinás M, Rickli EE
...
nmrlearner
Journal club
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08-22-2010 02:27 PM
[Nature network NMR forum] Journal club: structural biology of HIV infection (0 replies)
Journal club: structural biology of HIV infection (0 replies)
A recent paper in Science highlights once again the potential of NMR to yield insights into complex molecular recognition events in critical biological processes.
In this case the authors used a combination of X-ray crystallography and NMR to probe the structure of the extracellular terminus of the CCR5 co-receptor which, in concert with human CD4 and HIV gp120, is crucial for viral entry. The authors applied challenging saturation transfer effects to study the structure of a small synthetic peptide in a ternary...
nmrlearner
News from other NMR forums
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08-21-2010 03:29 PM
[Nature network NMR forum] Journal club: structure of the ternary Prp31:15.5K:U4 snRNA complex (1 reply)
Journal club: structure of the ternary Prp31:15.5K:U4 snRNA complex (1 reply)
I would like to start the “journal club” section of the NMR forum by presenting my favorite paper from the recent literature.
The paper (in this weeks issue of Science) is, in my opinion at least, a perfect example of the ability of NMR to contribute to the structural analysis of a challenging RNA-protein complex
I chose this high-impact paper because the combination of biochemistry, NMR, and crystallography on a ternary complex is how I would like to tackle a challenging project that I am...
Journal club: recombinant RNA expression (0 replies)
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