Highly efficient and easy protease-mediated protein purification
- 723 Downloads
As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate.
KeywordsProtein purification method Protease resistance Proteolysis Thermostability Site-specific cleavage NADH activity assay
We thank the Studienstiftung des Deutschen Volkes (Bonn, Germany) for a stipend to Daniel Last. Furthermore, the authors are grateful to Lilly Skalden for providing us with data on AspFum thermostability and Prof. Dr. Winfried Hinrichs and Dr. Gottfried Palm (all Institute of Biochemistry, Greifswald University) for the GFP expression construct.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Baumann M, Stürmer R, Bornscheuer UT (2001) A high-throughput-screening method for the identification of active and enantioselective hydrolases. Angew Chem Int Edit 40(22):4201–4204. doi: 10.1002/1521-3773(20011119)40:22<4201::aid-anie4201>3.0.co;2-v CrossRefGoogle Scholar
- Ericsson DJ, Kasrayan A, Johansson P, Bergfors T, Sandstrom AG, Backvall JE, Mowbray SL (2008) X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation. J Mol Biol 376(1):109–119. doi: 10.1016/j.jmb.2007.10.079 CrossRefPubMedGoogle Scholar
- Harmsen MM, van Solt CB, van Zijderveld-van Bemmel AM, Niewold TA, van Zijderveld FG (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol 72(3):544–551. doi: 10.1007/s00253-005-0300-7 CrossRefPubMedGoogle Scholar
- Keil B (1992) Specificity of proteolysis. Springer Verlag, Berlin HeidelbergGoogle Scholar
- Li Y (2011) Self-cleaving fusion tags for recombinant protein production. Biotechnol Lett 33(5):869--881. doi: 10.1007/s10529-011-0533-8
- Skalden L, Thomsen M, Höhne M, Bornscheuer UT, Hinrichs W (2015) Structural and biochemical characterization of the dual substrate recognition of the (R)-selective amine transaminase from Aspergillus fumigatus. FEBS J 282(2):407–415. doi: 10.1111/febs.13149
- Thomsen M, Skalden L, Palm GJ, Hohne M, Bornscheuer UT, Hinrichs W (2014) Crystallographic characterization of the (R)-selective amine transaminase from Aspergillus fumigatus. Acta Crystallogr D Biol Crystallogr 70(Pt 4):1086–1093. doi: 10.1107/s1399004714001084
- Wyss M, Pasamontes L, Friedlein A, Remy R, Tessier M, Kronenberger A, Middendorf A, Lehmann M, Schnoebelen L, Rothlisberger U, Kusznir E, Wahl G, Müller F, Lahm HW, Vogel K, van Loon AP (1999) Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance. Appl Environ Microbiol 65(2):359–366PubMedCentralPubMedGoogle Scholar