Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling. Expression with Pichia is available, though expertise and equipment requirements surpass E. coli culture. We developed a system utilizing Saccharomyces cerevisiae, [13C]-glucose, and [15N]-ammonium chloride with complexity comparable to E. coli. Here we report two vectors for expressing the crystallizable fragment (Fc) of IgG1 for secretion into the culture medium, utilizing the ADH2 or DDI2 promoters. We also report a strategy to optimize the expression yield using orthogonal Taguchi arrays. Lastly, we developed two different media formulations, a standard medium which provides 86â??92% 15N and 30% 13C incorporation into the polypeptide, or a rich medium which provides 98% 15N and 95% 13C incorporation as determined by mass spectrometry. This advance represents an expression and optimization strategy accessible to experimenters with the capability to grow and produce proteins for NMR-based experiments using E. coli.
Metabolic15N labeling of the N-glycosylated immunoglobulin G1 Fc with an engineered Saccharomyces cerevisiae strain
Metabolic15N labeling of the N-glycosylated immunoglobulin G1 Fc with an engineered Saccharomyces cerevisiae strain
Abstract
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...
nmrlearner
Journal club
0
07-09-2022 07:39 AM
[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,...
nmrlearner
Journal club
0
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...
nmrlearner
Journal club
0
02-22-2016 12:44 PM
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...
nmrlearner
Journal club
0
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)...
nmrlearner
Journal club
0
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...
nmrlearner
Journal club
0
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...
nmrlearner
Journal club
0
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...