The predominant protein expression host for NMR spectroscopy is Escherichia coli, however, it does not synthesize appropriate post-translation modifications required for mammalian protein function and is not ideal for expressing naturally secreted proteins that occupy an oxidative environment. Mammalian expression platforms can address these limitations; however, these are not amenable to cost-effective uniform 15Â*N labeling resulting from highly complex growth media requirements. Yeast expression platforms combine the simplicity of bacterial expression with the capabilities of mammalian platforms, however yeasts require optimization prior to isotope labeling. Yeast expression will benefit from methods to boost protein expression levels and developing labeling conditions to facilitate growth and high isotope incorporation within the target protein. In this work, we describe a novel platform based on the yeast Saccharomyces cerevisiae that simultaneously expresses the Kar2p chaperone and protein disulfide isomerase in the ER to facilitate the expression of secreted proteins. Furthermore, we developed a growth medium for uniform 15Â*N labeling. We recovered 2.2Â*mg/L of uniformly 15Â*N-labeled human immunoglobulin (Ig)G1 Fc domain with 90.6% 15Â*N labeling. NMR spectroscopy revealed a high degree of similarity between the yeast and mammalian-expressed IgG1 Fc domains. Furthermore, we were able to map the binding interaction between IgG1 Fc and the Z domain through chemical shift perturbations. This platform represents a novel cost-effective strategy for 15Â*N-labeled immunoglobulin fragments.
[NMR paper] High-resolution NMR structures of the domains of Saccharomyces cerevisiae Tho1.
High-resolution NMR structures of the domains of Saccharomyces cerevisiae Tho1.
Related Articles High-resolution NMR structures of the domains of Saccharomyces cerevisiae Tho1.
Acta Crystallogr F Struct Biol Commun. 2016 Jun 1;72(Pt 6):500-506
Authors: Jacobsen JO, Allen MD, Freund SM, Bycroft M
Abstract
THO is a multi-protein complex involved in the formation of messenger ribonuclear particles (mRNPs) by coupling transcription with mRNA processing and export. THO is thought to be formed from five subunits, Tho2p, Hpr1p, Tex1p,...
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06-16-2016 12:06 PM
[NMR paper] NMR assignments of mitochondrial cyclophilin Cpr3 from Saccharomyces cerevisiae.
NMR assignments of mitochondrial cyclophilin Cpr3 from Saccharomyces cerevisiae.
NMR assignments of mitochondrial cyclophilin Cpr3 from Saccharomyces cerevisiae.
Biomol NMR Assign. 2016 Feb 20;
Authors: Shukla VK, Singh JS, Trivedi D, Hosur RV, Kumar A
Abstract
Cyclophilins regulate protein folding, transport and signalling through catalysis of proline isomerization, and are ubiquitously expressed in both prokaryotes and eukaryotes. Cpr3 is the yeast mitochondrial cyclophilin and it is structurally and biophysically...
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02-22-2016 12:44 PM
Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain
Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain
Abstract This report describes a novel method for overexpression of 13C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly 13C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast...
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06-27-2011 04:30 AM
NMR solution structure of subunit E (fragment E(1-69)) of the Saccharomyces cerevisiae V (1)V (O) ATPase.
NMR solution structure of subunit E (fragment E(1-69)) of the Saccharomyces cerevisiae V (1)V (O) ATPase.
NMR solution structure of subunit E (fragment E(1-69)) of the Saccharomyces cerevisiae V (1)V (O) ATPase.
J Bioenerg Biomembr. 2011 Mar 12;
Authors: Rishikesan S, Thaker YR, Grüber G
The N-terminus of V-ATPase subunit E has been shown to associate with the subunits C, G and H, respectively. To understand the assembly of E with its neighboring subunits as well as its N-terminal structure, the N-terminal region, E(1-69), of the...
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03-15-2011 04:06 PM
[NMR paper] Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
Related Articles Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
Biochemistry. 2005 Sep 6;44(35):11795-810
Authors: Estephan R, Englander J, Arshava B, Samples KL, Becker JM, Naider F
The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339)...
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12-01-2010 06:56 PM
[NMR paper] The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged he
The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged helix-turn-helix fold as determined by NMR spectroscopy.
Related Articles The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged helix-turn-helix fold as determined by NMR spectroscopy.
Nucleic Acids Res. 2003 Dec 15;31(24):7199-207
Authors: Ono K, Kusano O, Shimotakahara S, Shimizu M, Yamazaki T, Shindo H
Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular...
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11-24-2010 09:16 PM
[NMR paper] Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)
Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR.
Related Articles Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR.
FEBS Lett. 2000 Sep 29;482(1-2):25-30
Authors: Szabo CM, Sanders LK, Le HC, Chien EY, Oldfield E
We have expressed -labeled Saccharomyces cerevisiae iso-1 cytochrome c C102T;K72A in Escherichia coli with a yield of 11...
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11-19-2010 08:29 PM
[NMR paper] NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a
NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.
Related Articles NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.
J Mol Biol. 1999 Aug 20;291(3):661-9
Authors: Evans SP, Bycroft M
In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small...