Expression of Lupinus luteus cDNA coding for PR10 protein in Escherichia coli: Purification of the recombinant protein for structural and functional studies

• Autorzy: Sikorski M. M.
• Języki publikacji: EN

Abstrakty

EN

The cDNA clones coding for two pathogenesis-related protein homologues of PR10 class, LlPR10.lA 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-se- ries: pET-3a and pET-15b, carrying the T7 promoter. Both plasmids with sub- cloned LlprlO.la gene were overexpressed in E. coli, strain BL21(DE3)pLysS. The recombinant L/PR10.1A protein, overproduced in bacterial cells trans­formed with the pET-3a/LlprlO.la 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)e LlPR10.lA protein overproducted in E. coli cells harbour­ing the pET-15b/LlprlO.la plasmid was purified by chromatography on Ni2+- charged His.Bind Resin. Western blot analysis with rabbit serum containing anti-£/PR10.1AN antibody revealed identical immunochemical properties of the two recombinant polypeptides and native LIPR10.1A 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 LIPR10.1A 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.

Słowa kluczowe

EN

cDNA clone; pathogenesis; immunochemical analysis; Lupinus luteus; purification; protein; bacteria; Escherichia coli; polymerase chain reaction; cDNA coding; recombinant protein; overexpression

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 1997
• Tom: 44
• Numer: 3
• Opis fizyczny: p.565-578,fig.

Twórcy

autor

Sikorski M. M.

• Polish Academy of Sciences, Z.Noskowskiego 12-14, 61-704 Poznan, Poland; E-mail: [e-mail]

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