Evidence for multimerization of neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors - PubMed (original) (raw)

Evidence for multimerization of neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors

D A Daines et al. J Bacteriol. 2000 Sep.

Abstract

Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205-212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.

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Figures

FIG. 1

Modified LexA-based system for investigating protein-protein interactions. The LexA-based system was based on that of Dmitrova et al. (6). A homodimerizing fusion expressed from one of the wild-type (wt) LexA DBD::MCS plasmids (pSR658, pSR660, or pSR662) will bind to the wild-type LexA operator and repress expression of lacZ in the chromosome of the reporter strain SU101. A heterodimerizing fusion, one subunit expressed from one of the wild-type LexA DBD::MCS plasmids and the other from one of the mutated (mut) LexA DBD::MCS plasmids (pSR659, pSR661, or pSR663), will bind to the hybrid LexA operator and repress expression of lacZ in the chromosome of the reporter strain SU202. The choice of plasmid MCS (A, B, or C) is made according to the reading frame of the desired fusion. Wild-type and mutated DBD correspond to LexA1–87WT and LexA1–87408, respectively (6).

FIG. 2

Immunoblot of LexA-Neu fusions. Full-length E. coli K1 region 2 genes were ligated into pSR658. An immunoblot of crude lysates of induced stationary-phase cultures probed with anti-LexA antiserum (Invitrogen) is shown. The calculated molecular masses in kilodaltons for the wild-type and LexA fusions, respectively, are in parentheses. Lane 1, LexA::NeuD (22.2, 31.8); lane 2, LexA::NeuC (43.1, 52.7); lane 3, LexA::NeuB (38.1, 47.7); lane 4, LexA::NeuA (45.8, 55.4). Molecular mass markers in kilodaltons appear at the left.

References

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Evidence for multimerization of neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors - PubMed (original) (raw)