Small acid soluble proteins for rapid spore identification.

Branda, Steven S. ; Lane, Todd W. ; VanderNoot, Victoria A. ; Jokerst, Amanda S.

Sandia National Laboratories

关键词: 99 General And Miscellaneous//Mathematics, Computing, And Information Science;    Bacillus;    Fluorescence;    Detection.;    59 Basic Biological Sciences;   

DOI  :  10.2172/984135
RP-ID  :  SAND2006-7860
RP-ID  :  AC04-94AL85000
RP-ID  :  984135

美国|英语

来源: UNT Digital Library

【 摘 要 】

This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.