Mammalian Expression of Isotopically Labeled Proteins for NMR Spectroscopy

Abstract

NMR spectroscopic characterization of biologically interesting proteins generally requires the incorporation of (15)N/(13)C and/or (2)H stable isotopes. While prokaryotic incorporation systems are regularly used, mammalian ones are not: of the nearly 9,000 NMR macromolecular structures currently deposited in the Protein Data Bank, only a handful (<0.5%) were solved with proteins expressed in mammalian systems. This low number of structures is largely a reflection of the difficulty in producing uniformly labeled, mammalian-expressed proteins. This is unfortunate, as many interesting proteins require mammalian cofactors, chaperons, or post-translational modifications such as N-linked glycosylation, and mammalian cells have the necessary machinery to produce them correctly. Here we describe recent advances in mammalian expression, including an efficient adenoviral vector-based system, for the production of isotopically enriched proteins. This system allows for the expression of mammalian proteins and their complexes, including proteins that require post-translational modifications. We describe how this system can produce isotopically labeled (15)N and (13)C post-translationally modified proteins, such as the outer domain of HIV-1 gp120, which has 15 sites of N-linked glycosylation. Selective amino-acid labeling is also described. These developments should reduce barriers to the determination of NMR structures with isotopically labeled proteins from mammalian expression systems.

Effective Isotope Labeling of Proteins in a Mammalian Expression System

“…Adenoviruses used in our expression system are double-stranded DNA viruses (Berk, 1986) with a deletion of the E1 gene that renders it replication defective (Aoki, Barker, Danthinne, Imperiale, & Nabel, 1999; Sastry, Bewley, & Kwong, 2012). Recombinant adenovirus 5 (rAd5) initially binds cell surface coxsackie-adenovirus receptor (CAR), followed by an interaction with cellular integrins and subsequent internalization via receptor-mediated endocytosis.…”

“…2. Once an optimal construct for the GOI is identified from the transient transfection study, the target gene is subcloned into pVRC1290 shuttle vector using suitable restriction sites such that the Kozak sequence, localization sequences, and purification tags are retained (Aoki et al, 1999; Sastry et al, 2012). Protein expression using this shuttle vector can once again be tested using small-scale transient transfection (McLellan et al, 2013) in HEK293 adherent cells or any other cell line of choice.…”

“…Upon plaque formation cells are harvested, the recombinant crude virus is isolated and protein expression of the desired gene tested by infecting A549 (CAR+) lung carcinoma cells. Once protein expression is confirmed, the crude virus preparation is used to infect low-passage HEK293 cells to produce recombinant adenovirus as described previously (Sastry et al, 2012). Briefly, HEK293 cells are seeded at 2.0 × 10 7 cells per 15-cm plate 24 h prior to infection.…”

“…Small-scale protein expression using the adenoviral expression system can be performed in six-well plates using A549 adherent cells as described previously (Sastry et al, 2012). Typically, cells are seeded at0.8 × 10 6 cell/well in a six-well plate on Day 1 in fresh Dulbecco’s minimal eagle medium (DMEM) containing 10% heat-inactivated-dialyzed fetal bovine serum (FBS), 1% penicillin/streptomycin.…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…Also, recent advances in isotope labeling by mammalian expression may facilitate future glycosylation analyses by NMR measurements (Sastry et al, 2012). Novel therapeutic approaches involving proteases employ glycan modifications and may be applicable to KLKs and their inhibitors, in particular to those present in skin.…”