INVESTIGADORES
GONZALEZ Daniel Hector
artículos
Título:
Expression of sunflower homeodomain containing proteins in Escherichia coli: Purification and functional studies
Autor/es:
PALENA, CLAUDIA M.; GONZALEZ, DANIEL H.; GUELMAN, SEBASTIÁN; CHAN, RAQUEL L.
Revista:
PROTEIN EXPRESSION AND PURIFICATION
Editorial:
ACADEMIC PRESS INC ELSEVIER SCIENCE
Referencias:
Año: 1998 vol. 13 p. 97 - 103
ISSN:
1046-5928
Resumen:
Complementary DNA sequences encoding different portions of two sunflower
homeodomain proteins were cloned in-frame in the expression vectors
pRSET and pGEX-3X. When introduced into competent Escherichia coil cells
and induced, the resulting plasmids directed the expression of large
amounts (5-10% of total cellular protein) of the encoded polypeptides.
As a rule, fusions in pRSET rendered insoluble proteins, while fusions
in pGEX were soluble and could be purified in a single step by selective
absorption onto glutathione-agarose beads, followed by elution with
free glutathione. The purified proteins showed both glutathione
S-transferase and DNA-binding activity, indicating that they retain
their native conformation. The expression-purification protocol that was
employed allowed the isolation of up to 0.7 mg of protein per gram of
transformed cells. One of the fusion proteins, RH11 (which is a fusion
of the homeodomain protein HAHR1 in pRSET), though insoluble, was able
to bind DNA when spotted onto a nitrocellulose filter. This protein
could also be simply purified in large amounts by electroelution from
sodium dodecyl sulfate-polyacrylamide gels and used to elicit antibodies
which recognized both the transgenic fusion and the native protein from
sunflower nuclei. Our results clearly show that vector choice is a
critical parameter for obtaining large amounts of a desired protein for
particular purposes.