(518h) Protein Purification Using Lytag and Q Sepharose

Protein
Purification using LYTAG and Q Sepharose

Gina L. Pietro, Michael J.
Coolbaugh, Miriam S. Tang, David
W. Wood

A
method of purifying recombinant proteins using affinity
chromatography with Q Sepharose and a LYTAG-intein affinity tag was
developed. The LYTAG protein affinity tag is currently used with a
specifically designed resin for recombinant protein purification.
Inteins are proteins that have been engineered to cleave on a shift
in pH or temperature, facilitating their use as a component of
self-cleaving tags. The gene that creates LYTAG is joined with the
gene for a self-cleaving intein, and this combined gene is joined to
the gene for an interchangeable product protein. The cell then reads
the genes and creates proteins, called fusion proteins, made of LYTAG
connected to intein and an interchangeable product protein to be
purified. The LYTAG-intein fusion proteins can be attached to the Q
Sepharose resin. The intein can then be forced to cleave leaving
pure product to be eluted from the column.

Q
Sepharose resin is usually used as an anion exchanging resin that
separates proteins based on charge. This method utilizes the special
affinity that LYTAG has to quaternary amines, with the controlled
cleaving of intein using pH shift and temperature change. Initially,
the LYTAG-intein fusion protein is bound to the Q Sepharose column
utilizing its specific affinity for quaternary amines rather than
ionic interaction. The fusion protein is bound to the column using
high salt concentrations to force off other proteins that might have
bound to the column with ionic interactions. Equilibrating the column
to a lower pH and raising the temperature induces intein
self-cleavage. The pure product can then be eluted from the column.
From qualitative analysis of SDS-PAGE gel, the purity of the product
protein appears to be high.

Because
of the ability of Q Sepharose to be scaled up, this technology could
mean lab scale research and drug development would more easily
translate to industry. It would also be a step toward
introducing intein technology to industry. This process could be
implemented in the bio-pharmaceutical industry using existing Q
Sepharose columns with minimal
regulatory approval or change to current setup.