Bernard Amegadzie

Proteolytic Activity of Human Osteoclast Cathepsin K EXPRESSION, PURIFICATION, ACTIVATION, AND SUBSTRATE IDENTIFICATION

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F. H., Dodds, R., James, I., Connor, J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M.(1996) J. Biol. Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (k/[I] = 273,000 M•s). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.

Identification, sequence, and expression of the gene encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase☆

The gene, rpo 132, encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase was identified and sequenced. Two complementary approaches, involving antiserum to purified vaccinia virus RNA polymerase, were used to locate the rpo 132 gene. One method involved the screening of a lambda gt11 library of vaccinia virus genome fragments and the other was based on the immunoprecipitation and polyacrylamide gel electrophoresis of the in vitro translation products of mRNA that hybridized to immobilized vaccinia virus DNA. The deduced open reading frame of the rpo 132 gene predicted a polypeptide of 1164 amino acid residues with sequence similarities to the second-largest RNA polymerase subunits of eubacteria, archaebacteria, and eukaryotes as well as to other poxviruses. Transcriptional analyses indicated that rpo 132 has both early and late RNA start sites and is expressed throughout infection.

Evaluating energetics of erythropoietin ligand binding to homodimerized receptor extracellular domains

Abstract A number of techniques have been employed to characterize the energetics of EPO-EPOR-Fc interactions. AUC studies have shown that EPO and EPOR-Fc exist as monomers at concentrations less that 10 μM. Under these conditions, EPO and the EPOR-Fc associate to form a 1:1 complex and this complex does not undergo any further assembly processes. Studies in which the biological activity of EPO at a cell surface is competed by free and dimerized receptor show that the dimerized receptor is 750-fold more potent. This suggests that EPO is bound by both receptor subunits on the Fc chimera, as shown in Fig. 9D. This assembly model provides a foundation for interpretation of the kinetic, thermodynamic, and spectral results. SPR kinetic analyses of the EPO-EPOR-Fc interaction yields association and dissociation rate constants of 8.0 × 10 7 M −1 sec −1 and 2.4 × 10 −4 sec −1 , respectively, for an overall affinity of 3 p M (see Fig. 12). The half-maximal response in a cellular proliferation assay is evoked at an EPO concentration of 10 p M , 54 which is similar to the affinity kinetically determined for the EPOR-Fc. This value suggests that the EPOR-Fc chimera may be a reasonable model for the receptor on a cell surface (see Fig. 17). The use of this reagent is also supported by the studies of Remy et al. , who demonstrate that the EPOR is likely to exist as a dimer on the cell surface, in the absence of ligand. Titration calorimetry confirms the 1:1 stoichiometry, observed by AUC and SPR approaches. Further, the temperature dependence of the enthalpy yields a heat capacity that can be interpreted in terms of a large conformational change in the EPOR on EPO binding. Comparing the structures of the free and complexed receptor, some conformational changes are noted in loops L3 and L6. 18 However, these changes are small compared with the conformational changes predicted from an analysis of the calorimetric data reported here, i.e., equivalent to the folding of ∼70 amino acids. The change in buried surface area between the free and complexed EPOR, determined from structural data, is also quite small when compared with the predicted value of 7500 A 2 from calorimetry. Further studies need to be done to rationalize these observations. These may include an attempt to determine if conformational changes are communicated to the Fc domain and the extent to which EPOR extracellular domains are oriented on the Fc domain in a manner that faithfully reflects their orientation on a cell surface. Finally, while changes in the CD spectra are observed on binding of EPO to the EPOR-Fc, and the monomeric receptor, these changes may be due to subtle changes in the microenvironments of tryptophans and tyrosines and do not require conformational changes of the magnitude suggested from the calorimetry results. In summary, to define macromolecular interactions in solution, the stoichiometry, thermodynamics, and kinetics of assembly need to be understood. This task requires that a multitechnology approach be implemented. Here, AUC established an assembly model and provided a foundation on which SPR, ITC, and CD studies could be based and from which interpretation of these data could be extended. SPR established that the affinity of the dimerized receptor was high and ITC suggested that there may be a significant conformational change on binding. CD suggested that observed spectral changes may be due to these presumed conformational changes, but would also be consistent with more subtle changes. These studies further demonstrate that the EPOR-Fc is a valid model for the dimerized receptor on the cell surface and, as such, will be a useful tool for probing the differences in the interactions of the receptor dimer with EPO agonists and antogonists.