Hi,
Balraj Doray wrote:
>> Hi all,
> I'm presently using the his-tag sequence from Novogen's pET19b which
> has 12 histidines(10 in tandem), 4 aspartates(enterokinase cleavage site), 2
> glycines, 2 serines, a methionine, a lysine and an isoleucine residue in the
[...]
> What I'm observing is this tag is making a vast difference to the
> folding/assembly characteristics of some of my mutant fusions, namely, the
> presence of the tag greatly facilitates assembly of my mutant proteins.
I would suspect that the tag just increases the solubility of your
protein.
With "facilitate" you mean that you get a better yield, don't you? The
yield of the original protein without tag might be reduced because it
has some tendency to aggregate. The Tag with 4 Asp makes it stay
soluble even at the high concentrations usually used for assembly
experiments. High must here be understood in the context of protein
folding, where one often works at concentrations of only some
mikrograms per milliliter to prevent aggregation.
In the same way, the tag might also slightly enhance the assembly
kinetics, in that it shifts the equilibrium between
association-competent monomers and (reversibly aggregated) wrong
oligomers to the former.
Frank
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Frank Fuerst, Institute of Biochemistry of Potsdam University
Im Biotechnologiepark, D-14943 Luckenwalde
Tel.: +49-3371-681334; Fax.: +49-3371-681339
ffrank at rz.uni-potsdam.de
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