Using E coli to make proteins

The development of recombinant protein expression has transformed our understanding of biomolecular systems and the ability to manipulate these for clinical purposes. In the past decades, many proteins have been produced in E. coli, because yields are higher and costs lower than in other hosts. This has allowed the investigation of protein structure, interactions with biological molecules and synthetic ligands, and the elucidation of their biological activities from the level of cells to that of tissues and the organism. Many of these protein studies were designed to explore the structures underpinning activities and interactions, which have then led to the screening and identification of small molecule inhibitors. For example, many small molecule kinase inhibitors (e.g., imatinib and dasatinib, which interact with the kinase domain of Abelson murine leukemia viral oncogene homolog (ABL)) are used to treat cancers (Zhang et al., 2009). Moreover, the proteins themselves may have activities that are medically useful, exemplified by the use of FGFs in treating wounds and various ulcerative conditions (Nunes et al., 2015). So, it is very important to efficiently produce soluble and stable proteins with corresponding biological activities.

Central to this thesis was the production of a series of FGFs with well characterised HS binding properties to investigate the functional significance of their interactions with the HS co-receptor. In Chapter 3, HaloTag was successfully used as a solubilisation partner to express FGFs. HaloTag enabled a number of FGFs that were

expressed as insoluble proteins to be soluble; moreover, the yield of proteins was often increased. Some FGFs were still hardly expressed as soluble proteins (e.g., Halo-FGF16) even with the HaloTag fusion. The yield of a protein is also an important issue, since a high yield of soluble protein makes purification simpler and also reduces the expense of protein production, which are important for both laboratory and large scale production of proteins. Thus, there is still considerable optimisation required for the expression of some of these FGFs. Another important aspect is the biological activities of the recombinant protein. The biological activities of produced FGFs were mainly tested by measuring the stimulation of MAPK phosphorylation. The cell signalling study indicates that the N-terminal HaloTag did not change the stimulation of phosphorylation of p44/42MAPK _{by the FGFs, which} suggests HaloTag has little effect on their biological activities. This is consistent with the fact that different FGFs possess N-terminal extensions that vary greatly in length (Fig. 1.6 and (Xu et al., 2012)) and with the observation that FGF2 with a gold nanoparticle of ~9 nm diameter conjugated to the N-terminus stimulated MAPK phosphorylation identically to native FGF2 (Duchesne et al., 2012). Since some proteins (Halo-FGF16, FGF17 and Halo-FGF17) did not cause obvious phosphorylation of p44/42MAPK, a cell growth assay was used to test their biological activity. Cell growth is regulated by both cell division and cell survival, which is a more conventional and straightforward way to test the bioactivities of FGFs. The positive effect on cell growth indicates that these three proteins are also correctly folded, but in these cells they may mediate their effects by signalling pathways other than phosphorylation of p44/42MAPK. These FGFs all bind to heparin, a property that depends on the correct folding of the protein, since the canonical heparin binding site is formed from amino acids that are distant in sequence. Indeed, a peptide

corresponding to the part of the canonical binding site of FGF2 that is contiguous in sequence has an affinity 1000-fold lower for heparin than FGF2 (Kinsella et al., 1998). Taken together with the biological activity of the FGFs measured here, these data indicate that the purified Halo-FGFs are correctly folded proteins. Whether HaloTag has any subtle effects on MAPK or other signalling pathways activated by FGFRs (Section 1.7) could be explored in the future by removal of the HaloTag with TEV protease.

For some Halo-FGFs, e.g., Halo-FGF6, Halo-FGF8 and Halo-FGF22, HaloTag cannot be removed, since the released FGF always aggregates when TEV protease was used to cleave FGF from HaloTag. Thus, the work presented in Chapter 3 represents a partial solution to the challenge of producing high quality soluble recombinant FGFs.

A common strategy to produce soluble recombinant FGFs is to truncate the N- and sometimes the C-termini, leaving the core β trefoil core. However, these parts of the FGFs are likely to have important functions for two reasons. Firstly, there is considerable variability in these regions of FGFs, which is most pronounced between FGF subfamilies, whereas within a subfamily these regions are of more similar length (Fig. 1.6 and (Xu et al., 2012)), suggesting they have undergone the same natural selection processes that led to the diversification of the FGF family and its functions. Secondly, the fact that they are largely unstructured itself also supports this contention (Beenken et al., 2012, Moy et al., 1996, Olsen et al., 2006). The unstructured regions of proteins are usually involved in molecular interactions, and are involved in the molecular regulation of signalling processes (Wright and Dyson, 2015); in the case of the FGFs, there is evidence that this includes binding to the

FGFR and to HS via HBS3 for that part of the N-terminal unstructured region closest to strand 1 (Olsen et al., 2006, Beenken et al., 2012, Uniewicz et al., 2010, Xu et al., 2012). Thus, it will be important to develop means to produce full length soluble FGFs, including the more difficult cases. In some instances, although yields are far lower, mammalian expression systems may have to be employed.

Since some FGF2 medicines have already been used to accelerate wound healing, the applications of FGF medicines are constantly being extended. As increasing numbers of studies are focused on the biological functions of different FGFs from embryonic development to tissue repair and some diseases, successful production of FGFs will be of great importance, which may lead us to new FGF medicines in the future.