Overproduction of Thermus sp. strain T2 β-galactosidase in Escherichia coli and preparation by using tailor-made metal chelate supports
2003
Pessela, Benevides C. | Vian, Alejandro | Mateo González, César | Fernández-Lafuente, Roberto | García, José Luis | Guisán, José Manuel | Carrascosa, Alfonso V. | Comunidad de Madrid | Centro Nacional de Investigação Científica (Angola)
A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.
Mostrar más [+] Menos [-]This work was supported by grant COR070/94 from the Comunidad Autonoma de Madrid. We thank Hispanagar SA for its kind financial support. Benevides C. C. Pessela was the recipient of a Ph.D. fellowship from the government of Angola.
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