EXPRESSION OF HUMAN CHORIONIC-GONADOTROPIN UNIFORMLY LABELED WITH NMRISOTOPES IN CHINESE-HAMSTER OVARY CELLS - AN ADVANCE TOWARD RAPID-DETERMINATION OF GLYCOPROTEIN STRUCTURES

Most secreted eukaryotic proteins are modified by glycosylation, and i t has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carboh ydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solve d by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycos ylated hCG is biologically inactive, the crystallographic structure re quires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature o f interactions between the carbohydrate side chains and the protein ba ckbone by crystallographic methods. Structural information via NMR tec hniques can be obtained from proteins in solution if they can be unifo rmly labeled with C-13 and N-15 isotopes. We report the first such uni form labeling of a glycoprotein using a universal C-13- and N-15-label ing medium to express C-13,N-15-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG a s well as that of its free subunits. The C-13,N-15-labeled recombinant hCG and its separated subunits are shown to be nearly identical to ur inary hCG reference preparations on the basis of protein chemical stud ies, immunochemistry, biological activity, and the capability of isola ted 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 s ubunit purification was that lyophilization of glycoproteins from trif luoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.