Related ArticlesProduction of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin ?v?6 binding peptides for NMR.
Mol Biosyst. 2010 Oct 18;
Authors: Wagstaff JL, Howard MJ, Williamson RA
The integrin ?v?6 is up-regulated in several cancers and has clinical potential for both tumour imaging and therapy. Peptide ligands have been developed which show good binding specificity for ?v?6 and provide an opportunity to study the interaction in more detail by NMR. Such studies ideally require (15)N and (13)C labelled peptides, and recombinant expression within E. coli provides a cost effective way of generating isotopically labelled proteins and peptides. In this study we have used an insoluble fusion partner (ketosteroid isomerase) to produce high yields of recombinant peptide. The insoluble nature of the fusion allowed simple product recovery by cell lysis and centrifugation, and thorough washing of the insoluble pellet to remove contaminating proteins avoided the need for nickel-affinity chromatography in denaturing conditions which is the standard procedure. The protocol described here is convenient to scale-up and requires only one chromatography step (reverse-phase HPLC) which is comparable to solid-phase synthesis.
PMID: 20953501 [PubMed - as supplied by publisher]
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.
J Biomol NMR. 2011 Jun 12;
Authors: Sastry M, Xu L, Georgiev IS, Bewley CA, Nabel GJ, Kwong PD
NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in (15)N, (13)C, or (2)H. The bacterial expression systems currently in use to...
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06-15-2011 01:15 PM
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy
Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy
Abstract NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an...
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06-15-2011 02:31 AM
Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells
Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells
Abstract The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed...
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01-09-2011 12:46 PM
[NMR paper] An efficient fusion expression system for protein and peptide overexpression in Esche
An efficient fusion expression system for protein and peptide overexpression in Escherichia coli and NMR sample preparation.
Related Articles An efficient fusion expression system for protein and peptide overexpression in Escherichia coli and NMR sample preparation.
Protein Pept Lett. 2003 Apr;10(2):175-81
Authors: Cheng Y, Liu D, Feng Y, Jing G
An efficient fusion expression system with a small fusion partner, His6-tagged N-terminal fragment of staphylococcal nuclease R, has been constructed and tested with two genes. The results show that...
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11-24-2010 09:01 PM
[NMR paper] Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipo
Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipolar coupling measurement in stretched polyacrylamide gel.
Related Articles Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipolar coupling measurement in stretched polyacrylamide gel.
J Am Chem Soc. 2002 Mar 20;124(11):2450-1
Authors: Chou JJ, Kaufman JD, Stahl SJ, Wingfield PT, Bax A
The structure of a water-insoluble fragment encompassing residues 282-304 of the HIV envelope protein gp41 is studied when solubilized by...
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11-24-2010 08:49 PM
[NMR paper] Analysis of a peptide inhibitor of paramyxovirus (NDV) fusion using biological assays
Analysis of a peptide inhibitor of paramyxovirus (NDV) fusion using biological assays, NMR, and molecular modeling.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Analysis of a peptide inhibitor of paramyxovirus (NDV) fusion using biological assays, NMR, and molecular modeling.
Virology. 1997 Nov 24;238(2):291-304
Authors: Young JK, Hicks RP, Wright GE, Morrison TG
To investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and...
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08-22-2010 05:08 PM
[NMR paper] Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and
Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.
Protein Expr Purif. 1999 Jun;16(1):144-51
Authors: Landrieu I, Casteels P, Odaert B, De Veylder L, Portetelle D, Lippens G, Van Montagu M, Inzé D
The CKS1At...
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08-21-2010 04:03 PM
Enhanced production and isotope enrichment of recombinant glycoproteins produced in c
Abstract NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant...
Production of recombinant isotopically labelled peptide by fusion to an insoluble par
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