Protein refolding via immobilisation on crystal surfaces

QD Chemistry

Davidson, Katrina Ann ; Cooper, Alan

University:University of Glasgow

Department:School of Chemistry

关键词: Protein, Folding, Lysozyme, Lipase, PCMC, immobilisation;   

Others  :  http://theses.gla.ac.uk/345/1/2008Davidson1PhD.pdf

来源: University of Glasgow

【 摘 要 】

Is it possible to find an easy, generic method for protein refolding? The preparation offunctionally active protein molecules from the unfolded state can be a difficult task.Although there are many well-established techniques for protein refolding, such asdilution, dialysis, chromatography and others, in many instances these methods can be timeconsuming and inefficient. A rapid, inexpensive and simple method for protein folding is amuch sought after technique.Proteins in the unfolded state (either inclusion bodies or unfolded by chemical or physicalmeans) are generally solubilised in solutions containing urea or guanidine hydrochloride.The removal of these molecules from the protein environment is commonly utilised as amethod for triggering refolding.A new method for the refolding of biomolecular species has been developed via theformation of Protein Coated Micro-crystals (PCMC). The formation of PCMC is arecently developed method for the immobilisation protein upon the surface of a watersolubleexcipient (salt, amino acid or sugar) via a co-precipitation reaction in a watermiscible organic solvent. These proteins can then be used as immobilised biocatalysts inboth the aqueous and organic phase.In the immobilisation of unfolded, solubilised protein, the solubilising agents (e.g. urea orguanidine hydrochloride) are removed from the protein environment as they are soluble inthe organic phase. The removal of these molecules initiates protein folding during the coprecipitationprocess.In the course of this project, a number of proteins were studied in order to observe theirbehaviour in this immobilisation and simultaneous folding process. Lysozyme was utilisedas it is an enzyme which is relatively simple to refold from the chemically unfolded stateby conventional methods such as dilution. Upon immobilisation of lysozyme from thechemically unfolded state, up to 92% of the activity of the native protein was regained.The enzyme lipase, which is notoriously difficult to fold, was also used to determine theefficiency of this method under more challenging conditions. Lipase immobilised from thechemically unfolded state was seen to regain up to 36 % of the activity of the nativeprotein.