OVERPRODUCTION AND PURIFICATION OF SULA FUSION PROTEIN IN ESCHERICHIA-COLI AND ITS DEGRADATION BY ION PROTEASE IN-VITRO

To overproduce extremely unstable SulA protein, which is the cell-divi sion inhibitor of Escherichia coli, we fused the sulA gene to the malt ose-binding protein (MBP) fusion vectors with or without the signal se quence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-S ulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-Sul A fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre- MBP-SulA fusion protein was used to study the degradation of SulA prot ein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-d ependent manner. This result provides direct evidence chat Lon proteas e plays an important role in the rapid degradation of SulA protein in cells.