(239a) Proteomic Study of High Vaccine DNA Producer

Plasmid DNA is historically perceived as a means for recombinant protein production.  However, increasingly it has become recognized as a therapeutic product in its own right.  With many DNA vaccine clinical trials now underway and some showing promise, large quantities of plasmid DNA could be needed in the future.  In addition to enabling the therapeutic applications, producing plasmid DNA at a high yield now could improve the throughput of basic experimentation that requires transfection as a step. 

The goal of the work underway is to develop a production platform that has high volumetric productivity of plasmid DNA when inexpensive and simple minimal media is used.  We previously reported the use of E. coli DH5α and a mutated plasmid that achieved copy numbers in excess of 10,000.  In this prior work, plasmid-containing cells were selected for by sucrose and one facet of the mutation scheme was to alter the origin of replication region in order to reduce RNA1/RNA2 interaction.  Apart from the high copy numbers, there were two interesting findings:  (i) the growth rate was not affected as much as one would expect and (ii) a drastic alteration to cell morphology occurred.

To resolve how the host adapts to the sizable burden imposed, proteomic and metabolic studies have commenced, and this presentation will discuss some of the findings.  The changes that occur in the relative abundance of many ribosomal proteins are of particular interest.  These changes suggest that adaptation may be occurring in the high DNA-producers at a more general level where ribosomal assembly and recycling is altered.  Such changes and others may also account for the altered cell morphology.  In addition to proteomics, the effect of different carbon sources on product yield will be discussed as well as the changes in metabolic enzymes and stress proteins that also correlate to more traditional measurements such as acetate analysis.