Experimental and theoretical analysis of the chromatographic behaviour of protein purification fusions carrying charged tails.
Poly(glutamic acid) tail consisting of 6 glutamate residues was fused to the N-terminus of Escherichia coli beta-galactosidase (beta-gal), by genetic engineering techniques. The wild-type and modified genes were expressed intracellularly and in soluble state in Escherichia coli, leading to the proteins respectively designated beta-gal2 and E6-beta-gal. Both enzymes were purified by affinity chromatography. The specific activity of purified E6-beta-gal was found to be comparable to the wild-type enzyme and its increased net charge was indicated by lon-Exchange Chromatography (IEC). The use of such a charged fusion for selective recovery of beta-gal from cell extract using IEC and Ion-Exchange Membrane Chromatography (IEMC) was explored. The additional charges enabled the separation factor to be increased about two-fold on both IEC and IEMC, but the IEMC step achieved a better throughput than the IEC step. The selectivity of recovery promoted by the charged tail was further analysed by processing the experimental data obtained in IEC with the Stoichiometric Displacement Model, a recent model very appropriate for the understanding of the retention of polymeric biomolecules on ion-exchangers. It was shown that E6-beta-gal had the same characteristic charge as beta-gal2 but that the binding constant to the ion-exchanger of the tagged beta-gal was 6 times greater than for the wild-type enzyme.
Le Borgne
S
S
Département de Génie Biochimique et Alimentaire, UA CNRS 544 INSA, Toulouse, France.
Graber
M
M
Condoret
J S
JS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004274:DNA, Recombinant / Q000235:genetics; D015195:Drug Design; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011099:Polyglutamic Acid / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000235:genetics
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D011099:Polyglutamic Acid; D001616:beta-Galactosidase