Novel approaches for the creation of artificial metalloenzymes

Artificial metalloenzymes can be defined as the fusion of chemo- and enzymatic catalysts by insertion of non-specific achiral catalytic moieties into the chiral environment of a protein cavity, thereby broadening the scope of both fields. Presently, the field of artificial metalloenzymes is at an exciting stage, with interesting features already demonstrated and a growing reaction repertoire, but has not yet reached maturity. To exploit the full potential of catalytic reactions using hybrid catalysts is far from trivial and as the ability to screen for novel properties remains limited. Albeit computational simulations are advanced, directed/designed evolution of such selective catalysts remains challenging, hence their development relies to a great extent on trial-and-error. Hitherto, screening and evaluation of ligands for activity or of proteins for selectivity still remains an elaborate process, as all library members have to be evaluated individually. High-throughput analysis to simultaneously evaluate large hybrid catalyst libraries within a short time-frame has therefore become increasingly important.
The key focuses of this research were the development of (i) novel purification strategies to simplify the challenge of producing functional protein (streptavidin), in sufficient quantity and appropriate purity for high-throughput screening of organometallic moieties; (ii) a reiterative approach to protein expression: re-design of streptavidin construct, moving to a more complex expression system, Pichia pastoris, to increase the production of streptavidin and simplify the purification process; and (iii) a novel biomolecular scaffold, human carbonic anhydrase II, for the creation of a new artificial metalloenzyme.
This thesis addresses the issues and feasibility of purification and expression processes and, highlights bottlenecks that arose using these technologies, key learning's and where to go from now.