Biomedical subjects

Katherine A Sacksteder

Publications and source records attributed to Katherine A Sacksteder.

3 recordsLinked to original sources

PubMed2006

The solution structure of antigen MPT64 from Mycobacterium tuberculosis defines a new family of beta-grasp proteins.

The MPT64 protein and its homologs form a highly conserved family of secreted proteins with unknown function that are found within the pathogenic Mycobacteria genus. The founding member of this family from Mycobacterium tuberculosis (MPT64 or protein Rv1980c) is expressed only when Mycobacteria cells are actively dividing. By virtue of this relatively unique expression profile, Rv1980c is currently under phase III clinical trials to evaluate its potential to replace tuberculin, or purified protein derivative, as the rapid diagnostic of choice for detection of active tuberculosis infection. We describe here the NMR solution structure of Rv1980c. This structure reveals a previously undescribed fold that is based upon a variation of a beta-grasp motif most commonly found in protein-protein interaction domains. Examination of this structure in conjunction with multiple sequence alignments of MPT64 homologs identifies a candidate ligand-binding site, which may help guide future studies of Rv1980c function. The work presented here also suggests structure-based approaches for increasing the antigenic potency of a Rv1980c-based diagnostic.

Amino Acid Motifs↗

PubMed2005

Design and optimization of a recombinant system for large-scale production of the MPT64 antigen from Mycobacterium tuberculosis.

Early clinical trials of a potential new tuberculosis (TB) diagnostic, the Patch Test for Active TB (PTAT), used MPB64 protein that was purified from the spent medium of Bacillus Calmette-Guérin (BCG) Tokyo 172 vaccine production. The yield was poor, 0.05 mg/L, and the process for purification of the protein was complex, requiring four chromatographic steps. The combination of yield and purification complexity compromised the ability to produce the PTAT diagnostic in quantities sufficient for larger clinical trials and commercialization. We report here a highly efficient method for the overexpression and purification of recombinant MPT64 from Escherichia coli (rMPT64) based upon a mild insolubility of rMPT64 following induction, and scalable anion-exchange and gel filtration chromatographies. Yields of protein were improved substantially to approximately 250 mg/L, and resulted in a preparation greater than 98% pure. Quantitative release assays were developed and used with MALDI-TOF mass spectrometry to confirm the identity of rMPT64. Using a guinea pig model of active TB, we found that rMPT64 elicited a specific immune response indistinguishable from that of MPB64 purified from BCG Tokyo culture filtrates. These results describe the first efficient and scalable protocol for production of rMPT64, demonstrate its activity in an animal model of active TB, and lay the foundation of ongoing and future use of the PTAT in clinical settings.

Amino Acid Sequence↗

PubMed2002

New tuberculosis vaccine development.

Tuberculosis (TB) is a devastating disease that kills more than three million people each year. Of these, 0.9 million are co-infected with HIV and numbers of infections and death continue to rise with the global spread of HIV. A new vaccine is desperately needed to control this epidemic that threatens to kill 90 million people over the next 3 decades. Outstanding work in research laboratories, combined with the success of genome sequencing, has resulted in a variety of candidate TB vaccines, many of which are sufficiently promising to advance into clinical trials. This review discusses the array of new candidate TB vaccines and the clinical studies that are currently planned.

Animals↗