Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C

Publikations-Art

Zeitschriftenbeitrag (peer-reviewed)

Autoren

Stressler, T.; Eisele, T.; Meyer, S.; Wangler, J.; Hug, T.; Lutz-Wahl, S.; Fischer, L.

Erscheinungsjahr

2016

Veröffentlicht in

Protein Expression and Purification

DOI

10.1016/j.pep.2015.09.024

The high specific lysyl endopeptidase (Lys-C; EC 3.4.21.50) is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functional properties. Up to now, the high price makes this application nearly impossible. In this work, the increased expression for Escherichia coli optimized Lys-C was investigated. The cloned sequence had a short artificial N-terminal pro-peptide (MGSK). The expression of MGSK-Lys-C was tested using three expression vectors and five E. coli host strains. The highest expression rate was obtained for the expression system consisting of the host strain E. coli JM109 and the rhamnose inducible expression vector pJOE. A Lys-C activity of 9,340 ± 555 nkat_Tos-GPK-pNA/L_culture could be achieved under optimized cultivation conditions after chemical refolding. Furthermore, the influence of the native pre-N-pro peptide of Lys-C from Lysobacter enzymogenes ssp. enzymogenes ATCC 27796 on Lys-C refolding was investigated. The pre-N-pro peptide was expressed recombinantly in E. coli JM109 using the pJOE expression vector. The optimal concentration of the pre-N-pro peptide in the refolding procedure was 100 µg/mL_{refolding buffer} and the Lys-C activity could be increased to 541,720 nkat_Tos-GPK-pNA/L_culture. With the results presented, the expensive lysyl endopeptidase can be produced in high activity and high amounts and the potential of Lys-C for tailor-made protein hydrolysates with bioactive (e.g. antihypertensive) and/or techno functional (e.g. foaming, emulsifying) properties can be investigated in future time studies.