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 lupin expression library of uninfected roots. The contribution of PR10 proteins to the overall mechanism of plant defence still remains unknown. In order to elucidate the structure and function of lupin PR10.1A protein, a substantial quantity of the protein was produced in an E. coli 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 the pET-3a/Llpr10.1a plasmid, was purified to homogeneity from the insoluble "inclusion bodies" by ammonium sulphate fractionation and two sequential chromatographic steps: ion-exchange chromatography on DE 52 cellulose followed by size exclusion chromatography on Superdex 75 FPLC column. The (His)6 LlPR10.1A protein overproduced in E. coli cells harbouring the pET-15b/Llpr10.1a plasmid was purified by chromatography on Ni2+-charged His. Bind Resin. Western blot analysis with rabbit serum containing anti-LlPR10.1AN antibody revealed identical immunochemical properties of the two recombinant polypeptides and native LlPR10.1A protein. The recombinant protein produced in pET-3a plasmid was renatured from its insoluble form, concentrated up to 22 mg/ml and submitted to crystallisation. However, the LlPR10.1A protein expressed in pET-15b plasmid precipitated from the solution when at a higher concentration (10 mg/ml). This preparation was used at a lower concentration as an antigen for the preparation of polyclonal antibodies for immunochemical studies.
Sikorski
M M
MM
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań. mmsik@ibch.poznan.pl
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; C053376:pathogenesis-related proteins, plant