Case Study: Enterokinase

The light chain of bovine enterokinase (EKL) is a serine protease that catalyzes the hydrolysis of peptide bonds in proteins. Enterokinase specifically cleaves after lysine in the consensus sequence Asp-Asp-Asp-Asp-Lys. The gene encoding the light chain of bovine enterokinase was codon-optimized and placed under the control of either the Tetrahymena metallothionein (MTT5) or starvation (SP1) promoter. Transformed cells were induced to express EKL and activity measured directly in spent culture medium (Figure 1). EKL was additionally purified from spent culture medium via ion exchange chromatography (Figure 2).

enterokinase-figure1
Figure 1. Comparison of MTT5 and SP1 induced expression of enterokinase. The light chain of bovine enterokinase was induced to express following addition of Cd2+ to the medium (MTT5) or following transfer of cells to starvation medium (SP1). Spent culture medium was assayed for enterokinase activity at various time points post-induction. Both MTT5 and SP1 induced expression leads to similar levels of extracellular enterokinase activity.
enterokinase-figure2
Figure 2. Purified recombinant bovine enterokinase. Tetrahymena cells were induced to express enterokinase. Spent culture medium was harvested and enterokinase was purified by ion exchange chromatography. Shown is purified enterokinase (2 ug) resolved by SDS-PAGE and detected by Coomassie stain.