Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli.

Biotechnol Prog

Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Apartado Postal 629, Irapuato, Gto. 36500, México.

Published: April 2005

AI Article Synopsis

  • DNA encoding a His-tagged 11S globulin from amaranth was expressed in two E. coli strains, resulting in different protein accumulation patterns; BL21 (DE3) produced mostly inclusion bodies while Origami (DE3) yielded soluble protein (76 mg/L).
  • Soluble proamarantin was found to assemble into trimers, and treatment with an enzyme revealed that it breaks down into acidic and basic chains, indicating proper protein folding similar to that in seeds.
  • The protein was successfully purified using a one-step technique, yielding 48 mg/L, which will help in further studies, including site-directed mutagenesis of this storage protein.

Article Abstract

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.

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http://dx.doi.org/10.1021/bp049923eDOI Listing

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Article Synopsis
  • DNA encoding a His-tagged 11S globulin from amaranth was expressed in two E. coli strains, resulting in different protein accumulation patterns; BL21 (DE3) produced mostly inclusion bodies while Origami (DE3) yielded soluble protein (76 mg/L).
  • Soluble proamarantin was found to assemble into trimers, and treatment with an enzyme revealed that it breaks down into acidic and basic chains, indicating proper protein folding similar to that in seeds.
  • The protein was successfully purified using a one-step technique, yielding 48 mg/L, which will help in further studies, including site-directed mutagenesis of this storage protein.
View Article and Find Full Text PDF

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