mobilis strains tested However, their respective PCNs were close

mobilis strains tested. However, their respective PCNs were closely matched within the same strain (Table 2). This indicated that the respective pUC18 or pACYC-184 derived plasmid backbones had little effect on their replication properties within Z. mobilis; which were primarily governed by the ca. 1,900 bp replicon fragment from pZMO7. Copy number was highest in the ATCC 29191 strain (ca. 20-40 plasmids per cell), and

considerably lower in the NCIMB 11163 and CU1 Rif2 strains (ca. 1-3 plasmids per cell). Further detailed studies will be required to establish the physiological basis for this inter-strain variation in PCN. Protein expression and proteomic applications within Z. mobilis To demonstrate a proof of principle, we selected a well-established glutathione/glutathione S-transferase (GST) affinity ‘pull-down’ approach [34] for use in Z. mobilis. In addition

to functioning as a convenient method for one-step protein isolation, TSA HDAC supplier (N-terminal) GST fusions have previously been shown to be beneficial for the expression of soluble (heterologous) proteins in bacteria [48]. The AcpP, KdsA, DnaJ, Hfq and HolC proteins selected as ‘bait’ were included in a previous proteomic study conducted in E. coli[35]. With the exception of the Hfq RNA chaperone [49], the respective properties of these proteins have not previously been analyzed in Z. mobilis. Four out of five proteins were expressed in a soluble form in both the ATCC 29191 and CU1 Rif2 strains, clearly demonstrating the effectiveness of the pZ7-GST vector-based system. The GST-HolC protein may have been expressed in an insoluble form, thus failing to be recovered in the (soluble) cell lysate fractions. Co-purifying proteins ADP ribosylation factor were identified for two of the four GST-fusion proteins that were expressed

in the ATCC 29191 strain (AcpP and KdsA). However, it should be noted that the plasmid-based GST-fusion protein expression is performed in a wild-type chromosomal background. Consequently, the GST-tagged bait proteins will be in direct competition with the corresponding endogenous bait proteins, for the capture of binding partners within the cell. Hence it may not be possible to capture and purify sufficient levels of interacting protein species to enable their subsequent detection or identification. The acyl carrier protein AcpP, which acts as a covalent carrier of fatty acid intermediates during their biosynthesis, co-purified with four other functionally-related enzymes.

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