Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures

Abstract

Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein backbone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C- and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

“…Thus this system may prove to be a viable system for both the recombinant expression of the glycoprotein hormone subunits and for the study of their folding during biosynthesis. Furthermore, as the Pichia pastoris system has been shown to be suitable for expression of single and dual 13 C-and 15 N-labeled proteins for nuclear magnetic resonance studies (Laroche et al 1994, Mason et al 1996, this system also has the potential of producing adequate amounts of isotopically labeled hCG and its isolated subunits for structural studies (Lustbader et al 1996, Weller et al 1996.…”

Looking into live cells with in-cell NMR spectroscopy

“…Amongst these, a few exotic approaches in mammalian CHO and HEK cells have been reported for NMR sample preparations (Hansen et al, 1992;Lustbader et al, 1996;Wyss et al, 1995). More prominent systems include the yeast Pichia pastoris and Baculovirus-infected insect cells (Chen et al, 2006;Pickford and O'Leary, 2004;Strauss et al, 2003Strauss et al, , 2005.…”

Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris

“…The O-glycans comprise mainly the convential saccharides (see above) [8,11,12]. CHO cells have also been used for generating 13 C/ 15 Nlabeled hCG for NMR studies [13]. Expression of hCG in baculovirus insect cell systems yields hCG which appears to have also oligomannose-type chains [14][15][16].…”

“…In the context of broader studies focused on the pharmaceutical use of P. pastoris expressed hCG (phCG) and on the use of P. pastoris cells to prepare 13 C/ 15 N-labeled phCG for three-dimensional studies of its structure in solution using NMR spectroscopy, a detailed glycan analysis of phCG was undertaken by mass spectrometry and NMR spectroscopy. In addition, attention was paid to possible changes in glycosylation pattern when changing the culturing conditions.…”