Rational design of enterokinase for the development of enhanced biopharmaceutical proteins

• Main Author: Pradhan, S.
• Published: University College London (University of London) 2013
• Subjects: 660.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626037

Enterokinase (EK) is a serine protease used to cleave therapeutic recombinant proteins during downstream processing. It has been selected for the activation and cleavage of a range of proprietary fusion proteins developed by Syntaxin Ltd. Whilst EK is well suited to this role in regards to substrate specificity, it has drawbacks, especially when it comes to expression in bacterial cells. Expression of EK in bacterial cells is the preferred expression method for process optimisation but is problematic due to its preference for inclusion body formation. This project describes efforts for improving the solubility of EK in E. coli using different constructs and mutagenesis. A total of four constructs were tested with two found to be soluble and one partially soluble. Two of the constructs (D4K-EK & pelB-EK) were found to readily form inclusion bodies (IB). Refolding of these constructs was undertaken and optimised using DoE. Only the refolded pelB EK showed significant activity, but refolded activity was found to vary greatly based on IB quality. The partially soluble pelB-EK construct exports to the periplasm for activation and soluble expression and was chosen for mutagenesis studies to improve soluble expression. A rational design approach using a range of sequence and structural bioinformatics methods including the consensus sequence, Hotpatch and statistical coupling analysis were utilised to identify fifteen stabilising mutants and seven mutants designed to increase surface charge. Of these potential mutants, ten (five stabilising, five surface charge) were created and analysed for activity, soluble yield and changes to secondary structure. Seven of the ten mutants showed measurable activity. Of interest were the surface charge mutants, which helped improve the purified yield by up to 2.5 fold. Also of note was consensus mutant V30Q which helped improve activity of periplasmic EK by 4.3 fold, whilst A32S and A44G visibly improved the thermo-tolerance of EK.