Project Description

Generation of a broad specificity nuclease by refolding of inclusion body expressed protein. Optimisation of downstream processes after cell lysis

At Peak Proteins we regularly design DNA constructs for clients, for insertion into mammalian, insect, and bacterial cell systems for over-expression of intracellular proteins. The expressed proteins then need to be isolated and purified to be supplied for a variety of uses determined by the client. Regardless of the chosen purification route, which will vary from protein to protein, the common starting point usually involves some form of cell lysis, which liberates the protein of interest from the cell, and takes us to the first step on the road to purification.

Cell lysis, by its nature, is a destructive process, tearing open the cells and breaking apart the carefully ordered cellular compartments and in doing so, liberating the protein, making it available for purification. Lysis is generally followed by a clarification step (centrifugation) to separate soluble protein (in the supernatant), from insoluble cell debris. However, cell lysis also releases a large amount of DNA and RNA into the supernatant, which often visibly exhibits itself as viscous mess, detrimentally affecting the subsequent clarification step, and even the downstream processing. Large viscous strands of DNA and RNA can potentially block chromatography columns, and compromise the chromatographic performance. Commercial sources of nuclease enzyme are available which can be added during lysis and effectively cleave these extended nucleotide chains. This reduces viscosity significantly, making lysis supernatants much easier to work with, and also improving isolation of the protein of interest. However, commercial nucleases are expensive particularly when working at larger scales.

Our Solutions

We decided to see if it was possible to express our own nuclease. The enzyme chosen is normally secreted, contains disulphide bonds and much of the literature on recombinant protocols focuses on secreting it from unusual microbial species which were not available to us. However we did notice a very old literature report that suggested it might be possible to make it in E.coli after expression in insoluble inclusion bodies. There were a number of challenges to this. The expression of an active nuclease would be expected to be toxic to the E.coli so we needed to try and force it all into inclusion bodies. Secondly having produced the inclusion bodies it is critical to refold the protein correctly including the generation of the two disulphide bonds. The expression was undertaken at 37oC and expression induced with a high concentration of IPTG both designed to force the inclusion bodies. Despite this, the cells were unhealthy and only reached low cell densities. However they clearly had expressed the protein which could be isolated in an inclusion body fraction. This was then solubilised and refolded in a buffer to enable correct disulphide bond formation. The refolded protein was then further purified from impurities and mis-folded material by a two step chromatographic process to yield a protein of >95% purity. Intact mass spectrometry on the final protein product showed that the construct was the expected mass less 4Da thereby demonstrating the correct formation of the two disulphide bonds. Not least, when tested for activity, the in-house enzyme was very active.

The Impact

We were able to express our own nuclease enzyme, as a tool protein for in-house use. Protein expression as inclusion bodies in E.coli allowed us to generate large quantities of protein which we were then able to refold and purify to a high purity. Analysis of the final product indicated that the protein was correctly folded, and this was confirmed when tested for nuclease activity. We now have a broad specificity nuclease, which makes cell lysis and clarification easier, and with fewer problems for downstream processing and chromatography, improving yields. We are able to produce this cheaply, and use at scale.


For further information or simply to say hello please contact us on 01625 238891 or email us