CHARACTERIZATION OF BASIC-PROTEINS FROM SPIROPLASMA-MELLIFERUM USING NOVEL IMMOBILIZED PH GRADIENTS

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has beco me the method of choice for efficient separation of complex protein mi xtures. Previously, analysis of the Spiroplasma melliferum proteome (p rotein complement of a genome) has been performed with pH 3-10 and nar row range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the res olution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gra dient of commercially available IPG ge l strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 a dditional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinyliden e difluoride (PVDF) membrane and analysed by N-terminal protein micros equencing. Protein spots with an initial peak yield of as little as 10 0 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstr ating the importance of improved sample handling procedures and analyt ical technologies. Many essential metabolic enzymes were shown to have basic pr, including: glyceraldehyde-3-phosphate dehydrogenase, pyruva te kinase, carbamate kinase and lactate dehydrogenase. A very basic pr otein (pI approximate to 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The adven t of novel basic (pH 6-11) IPGs has allowed the visualisation of a sig nificantly greater percentage of the 'functional proteome: that portio n of the total protein complement of a genome actively translated with in a specific time frame, on 2-D electrophoresis gels. This will aid i n the characterisation of translated gene products in conjunction with genome sequencing initiatives.