Enhanced Biosynthetically Directed Fractional Carbon-13 Enrichment of Proteins for Backbone NMR Assignments

Authors

Broc Wenrich, Bucknell UniversityFollow
Reilly Sonstrom, Bucknell UniversityFollow
Riju A. Gupta, Bucknell University
David Rovnyak, Bucknell UniversityFollow

Publication Date

2015

Description

Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or (13)C', etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%(w/w)u-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-(13)C glucose to expression media at induction, there is poor preservation of (13)Calpha-(13)Cbeta spin pairs in the amino acids ILV, leading to the absence of Cbeta signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.Copyright 2015 Elsevier Inc. All rights reserved.

Journal

Protein Expression and Purification

Volume

115

First Page

1

Last Page

10

Department

Chemistry

Link to Published Version

dx.doi.org/10.1016/j.pep.2015.08.005

DOI

10.1016/j.pep.2015.08.005

Recommended Citation

Wenrich, Broc; Sonstrom, Reilly; Gupta, Riju A.; and Rovnyak, David. "Enhanced Biosynthetically Directed Fractional Carbon-13 Enrichment of Proteins for Backbone NMR Assignments." Protein Expression and Purification (2015) : 1-10.