Mapping and identification of corynebacterium glutamicum proteins by two-dimensional gel electrophoresis and microsequencing

As a prerequisite for proteome analyses of Corynebacterium glutamicum separation of the cytoplasm and the membrane fraction was optimized and two- dimensional (2-D) gel electrophoresis was established. The resulting 2-D protein maps revealed over 1000 silver-stained protein spots separated by isoelectric point and molecular mass for cytoplasmic proteins and approximately 700 silver-stained spots for proteins of the membrane fraction. Proposing a mean size of 1 kbp per gene the complete C. glutamicum genome of 3 Mbp encodes 3000 different proteins; more than half of these can be located using the maps which are presently available. In this study 10 proteins were identified by N-terminal microsequencing, namely the 35 kDa antigen, antigen 84, ATP synthase subunits α, γ and δ, cysteine synthase, elongation factor G and Ts, enolase, and rotamase. For seven sequences, corresponding proteins could not be identified. Additionally, two proteins were specifically detected by immunoblotting, a corynebacterial porin and the cytoplasmic protein threonine dehydratase. The methods and 2-D maps established in this study will be the basis for comparative studies of protein expression and a detailed proteome analysis of C. glutamicum.