Crystal Contact Engineering Enables Efficient Capture and Purification of an Oxidoreductase by Technical Crystallization

Technical crystallization is an attractive method to purify recombinant proteins. However, it is rarely applied due to the limited crystallizability of many proteins. To overcome this limitation, single amino acid exchanges were rationally introduced to enhance intermolecular interactions at the crystal contacts of the industrially relevant biocatalyst Lactobacillus brevis alcohol dehydrogenase (LbADH). The wildtype (WT) and the best crystallizing and enzymatically active LbADH mutants K32A, D54F, Q126H, and T102E were produced with E. coli and subsequently crystallized from cell lysate in stirred-tank crystallizers. Notwithstanding the high host cell protein (HCP) concentrations in the lysate, all mutants crystallized significantly faster than the WT. Combinations of mutations resulted in double mutants with faster crystallization kinetics than the respective single mutants, demonstrating a synergetic effect. We observed the almost entire depletion of the soluble LbADH fraction at crystallization equilibrium, proving high yields. The HCP concentration was reduced to below 0.5% after crystal dissolution and recrystallization, and thus a 100-fold HCP reduction was achieved after two successive crystallization steps. The combination of fast kinetics, high yields, and high target protein purity highlights the potential of crystal contact engineering to transform technical crystallization into an efficient protein capture and purification step in biotechnological downstream processes. This article is protected by copyright. All rights reserved.