CHARGED FUSIONS FOR BETA-GALACTOSIDASE RETENTION IN ANION-EXCHANGE CHROMATOGRAPHY

The retention behavior of a series of negatively charged beta-galactos idase fusion proteins carrying an additional 1 (designated BGCD1), 5 ( BGCD5), 11 (BGCD11), and 16 (BGCD16) aspartate residues was studied. T he added tails promoted protein retention in order of tail length and were most effective closer to the isoelectric point. The two parameter s Z and I, obtained from the stoichiometric displacement model, were u sed to characterize the extent of binding between the protein and the ion-exchange surface. At pH 5.7, the Z number increased with tail leng th (charge) and was 11.5, 8.5, 6.9 and 5.3 for BGCD11, BGCD5, BGCD1 an d wild type beta-galactosidase (BGWT), respectively. At these conditio ns, the fusions had very similar I values which were five times smalle r than that of BGWT. However, the increase in Z numbers outweighed the decrease in I values and retention was enhanced. When BGWT was brough t to the same net charge (by increasing the mobile phase pH) as each o f the fusions, the Z number was similar to that of the corresponding f usion. However, the I value decreased with increasing pH (net charge) and was lower than that of the corresponding fusion by factors of 25-5 00. Consequently, despite the similar Z numbers, the fusions still had higher retention.