Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli.
DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coli is similar to that taking place in seeds. The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.
Medina-Godoy
Sergio
S
Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Apartado Postal 629, Irapuato, Gto. 36500, México.
Nielsen
Niels C
NC
Paredes-López
Octavio
O
D000485:Allergens; D027721:Amaranthus / Q000235:genetics* / Q000378:metabolism*; D052179:Antigens, Plant; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055314:Seed Storage Proteins; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013045:Species Specificity
D000485:Allergens; D052179:Antigens, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; C059777:cruciferin protein, Brassica napus; D006639:Histidine