Effective Isotope Labeling of Proteins in a Mammalian Expression System

Abstract

Isotope labeling of biologically interesting proteins is a prerequisite for structural and dynamics studies by NMR spectroscopy. Many of these proteins require mammalian cofactors, chaperons, or posttranslational modifications such as myristoylation, glypiation, disulfide bond formation, or N- or O-linked glycosylation; and mammalian cells have the necessary machinery to produce them in their functional forms. Here, we describe recent advances in mammalian expression, including an efficient adenoviral vector-based system, for the production of isotopically labeled proteins. This system enables expression of mammalian proteins and their complexes, including proteins that require posttranslational modifications. We describe a roadmap to produce isotopically labeled 15N and 13C posttranslationally modified proteins, such as the outer domain of HIV-1 gp120, which has four disulfide bonds and 15 potential sites of N-linked glycosylation. These methods should allow NMR spectroscopic analysis of the structure and function of posttranslationally modified and secreted, cytoplasmic, or membrane-bound proteins.

Insights from NMR Spectroscopy into the Conformational Properties of Man‐9 and Its Recognition by Two HIV Binding Proteins

“…We infected A549 lung carcinoma cells with an adenovirus construct encoding HIV‐1 gp120 . To increase the amount of Man‐9, cells were treated with 50 mg L −1 kifunensine (an α1‐2 mannosidase inhibitor that leads to increased production of Man‐8 and Man‐9), and gp120 was harvested by affinity and size‐exclusion chromatography . N‐Linked oligomannosides were enzymatically released from gp120 by using peptide‐ N ‐glycosidase F and purified by size‐exclusion chromatography followed by HPLC.…”

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…Presenting all the refined aspects of this topic is beyond the scope of our review. Still, we can mention some recent references focusing on the recombinant production of proteins dedicated to NMR analysis: i) for a uniform 2 H-, 13 C and/or 15 N-labeling in E. coli, [91][92][93][94][95] yeast, 96,97 insect cells, [98][99][100][101] or mammalian cells, 102,103 ii) for 13 C-methyl-labeling in E. coli, 104,85,105 yeast [106][107][108] or insect cells, [109][110][111] iii) for amino acid specific labeling in E. coli, [112][113][114] insect cells, 115 iv) for unnatural amino acids labeling containing 19 F-moieites [116][117][118][119] or other non-natural chemical functions. 119,120 The so-called cell-free approaches also offer a range of isotope labeling possibilities.…”

NMR Resolution Enhancement and Homonuclear Decoupling Using Non-Uniform Weighted Sampling

“…is either not sufficient, or not available, for example in mammalian expressed proteins 20 , or studies at natural isotopic abundance 21 ; increasing the accuracy of frequency-based measurements of residual dipolar couplings; combination with homonuclear decoupling in the direct dimension and/or NUS or spectral aliasing to obtain ultra high-resolution 2D measurements 2,22 ; sensitive and high-resolution analyses of the interactions of side-chains through titration experiments using uniformly 13C-labelled material; and as a simple approach to decouple Cα-Cβ scalar couplings in HNCA and other 3D experiments.…”