Expression of actin in Escherichia coli, its preparation in a soluble form, and its functional characterization
Frankel, Stewart A.
MetadataShow full item record
Bacterial strains were constructed for the expression of wild type and mutant Dictyostelium actin, and were shown to accumulate the appropriately sized actin species. These strains also accumulated a truncated 29 kDa actin species, which was shown to be the product of internal translation initiation. After the usual forms of bacterial lysis, both truncated and full-length actin were present in a highly insoluble form.;In order to better understand the nature and generation of insoluble protein, two bacterial strains were examined for the presence of morphological inclusions. The one expressing insoluble protein (actin) contained inclusions, but so did the one expressing soluble protein (a fragment of the myosin tail region). This latter finding was inconsistent with the hypothesis that inclusions were composed of insoluble protein and that insolubility was generated prior to lysis. Several lines of evidence pointed to an alternative hypothesis for the formation of highly insoluble actin: co-aggregation of the expressed protein with bacterial outer membrane components. Our hypothesis led to the development of two methods which yielded soluble, non-denatured actin: (1) the treatment of co-aggregates with Sarkosyl detergent, leading to a differential solubilization of actin, (2) lysis of the bacteria in the presence of Sarkosyl. Our procedures may prove useful for other expressed proteins which are normally soluble but become insoluble after bacterial expression.;Sarkosyl lysis was used as the starting point for several methods of purification. Wild type 42 kDa actin could be partially purified by anion exchange chromatography and gel filtration, or fully purified using DNase I affinity chromatography and gel filtration in 0.8 M NaCl. 42 kDa actin reversibly polymerized into filaments of the appropriate diameters, and bound myosin S-1 in an ATP-sensitive manner.;29 kDa actin had the following properties: (1) self-aggregation in a concentration-dependent manner, (2) association with expressed 42 kDa actin, a process largely inhibited by 0.8 M NaCl, (3) high affinity binding to DNase I.
- Theses and Dissertations