| Autor: |
Nowotny, Phillip, Hermann, Johannes, Li, Jianing, Krautenbacher, Angela, Klöpfer, Kai, Hekmat, Dariusch, Weuster-Botz, Dirk |
| Zdroj: |
Crystal Growth & Design; March 2019, Vol. 19 Issue: 4 p2380-2387, 8p |
| Abstrakt: |
Technical protein crystallization is an alternative to preparative chromatography for purification of proteins. However, only a few proteins are satisfactorily crystallizable for this technical purpose. In the present work, the crystallizability of Lactobacillus brevisalcohol dehydrogenase (LbADH) was significantly improved by rational engineering of its crystal contact patches. The concept was to exchange amino acids at the crystal contact patches with the objective of (i) surface entropy reduction (SER) and (ii) enhancement of ionic interactions. We present three newly designed, enzymatically active LbADH mutants with improved crystallizability: K32A (via SER) and Q126H and Q126K (both via enhancement of ionic interactions). The wild type crystallized with a low crystallization success rate in microbatch experiments. All mutants crystallized consistently with enhanced crystallization kinetics under identical conditions. Mutant K32A crystallized at reduced protein concentrations. Mutant Q126H crystallized at reduced concentrations of protein and the crystallization agent polyethylene glycol. Furthermore, the X-ray structure of mutant K32A reveals evidence of crystal contact enforcement which does explain enhanced crystallizability on the atomic level. The increased space–time yield of mutant K32A in stirred tank crystallizers demonstrates that rational crystal contact engineering is a powerful tool to promote technical protein crystallization. |
| Databáze: |
Supplemental Index |
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