Comparative studies on chemically and enzymatically coupled DNA-Sepharose columns for purification of a lac repressor...

The length of a DNA sequence attached to an affinity chromatography column affects column retention of transcription factors. Even when unrelated sequences such as a poly(A):poly(T) tail are included in a DNA sequence, transcription factors such as the lac repressor are bound more tightly by the column. The position of the additional sequences is also important. To compare coupling procedures, an identical DNA sequence was covalently attached to Sepharose by chemical coupling or produced enzymatically by template driven enzymatic primer extension. These two types of supports, containing the O1 operator sequence bound by lac repressor, were packed into identical columns and compared by purification of a lac repressor-beta-galactosidase fusion protein. We found that the purity and yield of proteins eluted from the two columns were similar. Overall, the results suggest that there is no significant advantage to either type of support for the purification of some proteins. The study revealed a potentially important effect of the length of DNA sequences on column selectivity.