EXPRESSION OF LUPINUS-LUTEUS CDNA CODING FOR PR10 PROTEIN IN ESCHERICHIA-COLI - PURIFICATION OF THE RECOMBINANT PROTEIN FOR STRUCTURAL AND FUNCTIONAL-STUDIES

The cDNA clones coding for two pathogenesis-related protein homologues of PR10 class, LlPR10.1A and LlPR10.1B, were identified in yellow lup in expression library of uninfected roots. The contribution of PR10 pr oteins to the overall mechanism of plant defence still remains unknown . In order to elucidate the structure and function of lupin PR10.1A pr otein, a substantial quantity of the protein was produced in an E. col i expression system using plasmids of pET-series: pET-3a and pET-15b, carrying the T7 promoter. Both plasmids with subcloned Llpr10.1a gene were overexpressed in E. coli, strain BL21(DE3)pLysS. The recombinant LlPR10.1A protein, overproduced in bacterial cells transformed with th e pET-3a/Llpr10.1a plasmid, was purified to homogeneity from the insol uble ''inclusion bodies'' by ammonium sulphate fractionation and two s equential chromatographic steps: ion-exchange chromatography on DE 52 cellulose followed by size exclusion chromatography on Superdex 75 FPL C column. The (His)(6) LlPR10.1A protein overproducted in E. coli cell s harbouring the pET-15b/Llpr10.1a plasmid was purified by chromatogra phy on Ni2+-charged His.Bind Besin. Western blot analysis with rabbit serum containing anti-LIPR10.1A(N) antibody revealed identical immunoc hemical properties of the two recombinant polypeptides and native LlPB 10.1A protein. The recombinant protein produced in pET-3a plasmid was renatured from its insoluble form, concentrated up to 22 mg!ml and sub mitted to crystallisation. However, the LlPR10.1A protein expressed in pET-15b plasmid precipitated from the solution when at a higher conce ntration (10 mg/ml). This preparation was used at a lower concentratio n as an antigen for the preparation of polyclonal antibodies for immun ochemical studies.