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An efficient on-column expressed protein ligation strategy: Application to segmental triple labeling of human apolipoprotein E3

¹ Department of Chemistry, School of Science, Tianjin University, Tianjin, China 300072
² Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA
³ Department of Chemistry, School of Science, Tianjin University of Science and technology, Tianjin, China 300222

(RECEIVED December 4, 2007; FINAL REVISION January 9, 2008; ACCEPTED January 10, 2008)

Expressed protein ligation (EPL) is an intein-based approach that has been used for protein engineering and biophysical studies of protein structures. One major problem of the EPL is the low yield of final ligation product, primarily due to the complex procedure of the EPL, preventing EPL from gaining popularity in the research community. Here we report an efficient on-column EPL strategy, which focuses on enhancing the expression level of the intein-fusion protein that generates thioester for the EPL. We applied this EPL strategy to human apolipoprotein E (apoE) and routinely obtained 25–30 mg segmental, triple-labeled apoE from 1-L cell culture. The approaches reported here are general approaches that are not specific for apoE, thus providing a general strategy for a highly efficient EPL. In addition, we also report an isotopic labeling scheme that double-labels one domain and keeps the other domain of apoE deuterated. Such an isotopic labeling scheme can only be achieved using the EPL strategy. Our data indicated that the segmental triple-labeled apoEs using this labeling scheme produced high-quality, simplified NMR spectra, facilitating NMR spectral assignment. For large proteins, such as apoE, perdeuterated protein samples have to be used to reduce the linewidth of NMR signals, causing a major problem for the NOE-based NMR method, since perdeuterated proteins lack protons for NOE measurement. The new labeling strategy solves this problem and provides ¹³C/¹⁵N double-labeled, protonated protein domains, allowing for determination of high-resolution NMR structure of these large proteins.

Keywords: expressed protein ligation; high-level protein expression; segmental isotope labeling; apolipoprotein E; NMR spectroscopy; nuclear Overhauser enhancement; NMR structure determination

Reprint requests to: Qianqian Li, Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA; e-mail: qil{at}med.wayne.edu; fax: (313) 577-8836; or Jianjun Wang, School of Medicine, Wayne State University, Room 5113, Scott Hall, 540 East Canfield Avenue, Detroit, MI 48201, USA; e-mail: jjwang{at}med.wayne.edu; fax: (313) 577-8836.

Abbreviations and symbols: apoE, apolipoprotein E; apoEC-J, the monomeric mutant of apoE C-terminal domain; apoE-J, the monomeric apoE mutant; CBD, chitin-binding domain; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; DTT, dithiothreitol; EDTA, ethylene-diamine-tetra-acetic acid; EPL, expressed protein ligation; HSQC, heteronuclear single quantum coherence; IPTG, isopropyl-beta-D-thiogalactopyranoside; LDL, low-density lipoprotein; MESNA, 2-mercaptoethanesulfonic acid; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCEP, tris[2-carboxyethyl]-phosphine; TROSY, transverse relaxation-optimized spectroscopy.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073383708.

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