Dr. David Calhoun conducts research projects that focus on regulation of the ilvGMEDA gene cluster of Escherichia coli K-12, and on enzyme replacement therapy for patients with Fabry disease. He is a member of Area Group II, and the Executive Committee of RCMl.
RESEARCH
Fabry Disease Research Link
* Expression of Glycosylated and Catalytically Active Recombinant Human a-Galactosidase A Using a Baculovirus Vector.
Mutations in the gene for the human lysosomal enzyme a-galactosidase A (EC 3.2.1.22) result in the sphingolipidosis called Fabry disease. The enzymatic defect is inherited as an x-linked recessive disorder and is associated with a progressive deposition of the glycosphingolipid substrates of the defective enzyme, including globotrioaslyceramide, galabioasylceramide, and blood group B substance. In affected males the deposition of these compounds occurs primarily in lysosomes of vascular endothelial and muscle cells, and leads to early demise due to occulusive disease of the heart, kidney, and brain. Attempts have been made in vivo and in vitro to replace the defective enzyme with normal enzyme obtained from various human sources. The lack of sufficient quantities of purified human a-galactosidase A has prevented a complete evaluation of the potential efficacy of enzyme replacement in Fabry disease.
We isolated a-galactosidase A specific cDNA and genomic clones and expressed the cDNA clones using the baculovirus, Autographa californica multinuclear polyhedrosis virus. In insect cells the baculovirus vector uses many of the protein modification, processing, and transport systems that also occur in vertebrae cells, including glycosylation. Thus, we wished to determine if sufficient human a-galactosidase A could be produced using this system for both structural analyses and therapeutic evaluations. We constructed baculovirus vectors that produces high levels of the human a-galactosidase A. The recombinant enzyme was purified from the culture medium of infected Sf9 cells and analyzed for the presence of N-linked carbohydrate and for uptake by normal and Fabry fibroblasts in culture. At present, collaborations with Enzon, Inc. are directed towards clinical trails using the recombinant a-galactosidase A produced in insect cells.
* Multiple Transcripts Encoded by the ilvGMEDA Gene Cluster of Escherichia coli K-12.
The ilvGMEDA cluster encodes five gene products needed for the biosynthesis of leucine, isoleucine, and valine in Escherichia coli K-12. Three promoters, ilvGp2, ilvEp, ilvAp, have been well characterized, and these initiate transcrpition just upstream of the ilvG, ilvE, and ilvA genes, respectively. The presence of the two internal promoters, ilvEp and ilvDp, was first inferred by using strains with polar insertions or mutations located the ilvGMEDA cluster, lilv phage carrying only ilvEDA, ilvDA, or ilvA, and fusions to reporter genes. The internal promoter, ilvAp, is preferentially expressed in cells grown under anaerobic conditions. A site of transcription termination has been characterized downstream of the ilva gene.
Northern blot hybridization was used to determine the identities of the mRNA species coded in vivo by the ilvGMEDA cluster. As probes we used synthetic oligonucleotides and asymmetric PCR products. Transcripts of 6.7, 4.6, and 1.1 kb were detected with probe oliE, whereas transcripts of 6.7, 4.6, and 3.6 kb transcripts, were not always detectable on short exposures of the blots to film or at low levels of expression. The 6.7, 4.6, 3.6, and 1.1 kb transcripts appeared to change in parallel in response to amino acid levels. These Northern blots represent steady state mRNA levels, and we have not yet measured thes separate rates of synthesis and degradation of each transcript under these growth conditions.
Selected Publications:
Kalyankar, N. D., Sharma, M. K., Vaidya, S., Calhoun, D. H., Maldarelli, C, Couzis, A., and Gilchrist, -L. (2006) Arraying of Intact Liposomes Into Chemically Functionalized Microwell Surfaces, Lang Muir (in press)*.
Chen, Y, M. Jin, L. Goodrich, G. Smith, G. Coppola, and D. H. Calhoun (2000) Purification and characterization of human a-galactosidase A expressed in insect cells using a baculovirus vector. Protein Expression and Purification, 20: 228-236.
Chen, Y, M. Jin, T. Egborge, G. Coppola, J. Andre, and D. H. Calhoun (2000) Expression and Characterization of Glycosylated and Catalytically Active Recombinant Human a-galactosidase A Produced in Pichia pastoris. Protein Expression and Purification, 20: 472-484.
Calhoun, D. H., C. A. Bonner, W. Gu, G. Xie and R. A. Jensen (2001) The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa, Genome Biology, 2:0030.1-0030.16.
Jensen, R. A., G. Xie, D. H. Calhoun, and C. A. Bonner (2002) The correct phylogenetic relationship of KdsA (3-Deoxy-D-manno-octulosonate 8-phosphate synthase) with one of two independently evolved classes of AroA (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase), J. Mol. Evol. 54: 416-423).
Hubbard, K., DeJesus, V., C. Davis, Y. Chen, D.H. Calhoun, and Z. Zakeri (2002) Cellular aging and lysosomal expression during cell death, Experimental Cell Research 274: 92-99.
Jin, M., Rosario, W, Watler, E., and D. H. Calhoun (2004) Development of a large-scale HPLC-based purification for the urease from Staphylococcus leei and determination of subunit structure, Protein Expression and Purification, 34: 111-117.
Location and Contact Information:
Dr. David Calhoun
Department of Chemistry, Room J1024
City College of New York
138th Street & Convent Avenue
New York, NY 10031
t. 212.650.6934
calhoun@scisun.sci.ccny.cuny.edu
Other Locations: J1309, J1310, and J1312.