Bacterial expression of biologically active recombinant musarmin 1 from bulbs of Muscari armeniacum L. and Miller.
Musarmins are type 1 ribosome-inactivating proteins with N-glycosidase activity on the 28 S rRNA that are present in bulbs of Muscari armeniacum L. and Miller at rather low concentrations. In the present work, a cDNA fragment coding for musarmin 1 was sub-cloned and expressed in Escherichia coli. The recombinant protein (rMU1) was synthesised as a polypeptide of 295 amino acids that was delivered to the periplasm and processed. Recombinant musarmin 1 present in the periplam has two forms: insoluble with a molecular mass of 29,423 and soluble with a molecular mass of 29,117 because of a small proteolytic shortening with respect to the insoluble one, presumably in the C-terminal. The yield of protein homogeneous by polyacrylamide gel electrophoresis was 23mgl-1 of bacterial culture. The recombinant musarmin 1 forms isolated from both the soluble and the insoluble (upon refolding) fractions retained full translational inhibitory and 28 S rRNA N-glycosidase activities as compared with the native protein. The recombinant protein displayed great stability towards trypsin, collagenase, rat plasma and rat liver protein extract, but was sensitive to the action of papain and proteinase K. The easy availability and full activity of the recombinant musarmin 1 makes it a good candidate for the preparation of immunotoxins for targeted therapy and for the construction of transgenic plants expressing it as antipathogenic agent.
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Valladolid, 47005 Valladolid, Spain.
Pilar
P
Antolín
Alessandra
A
Girotti
Francisco Javier
FJ
Arias
Begoña
B
Barriuso
Pilar
P
Jiménez
Ma Angeles
MA
Rojo
Tomás
T
Girbés
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D019668:Liliaceae / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000235:genetics*; D018517:Plant Roots / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity
D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; C494106:musarmin 1, Muscari armeniacum