Abstract
The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Measurement of phosphorylation of p42/44 mitogen-activated protein kinase and of cell growth demonstrated that the HaloTag fusion proteins were biologically active. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins.
Highlights
Of the 18 receptor-binding fibroblast growth factors (FGF), 15 bind a heparan sulfate co-receptor and are classed as growth factors and morphogens
Materials pET-14b vectors containing cDNAs encoding FGF1 and FGF2 and pET-M11 vector containing FGF7 cDNA were as described (Xu et al, 2012); cDNAs encoding FGF3, FGF10, FGF16, FGF17 and FGF20 were purchased from Eurofins Genomics (Ebersberg, Germany); cDNA encoding FGF6, FGF8 and FGF22 were purchased from Life Technologies (Paisley, UK); cDNAs encoding HaloTag was acquired from Kazusa DNA Research Institute (Kisarazu, Japan); Primers for PCR were from Life Technologies (Paisley, UK)
Expression of soluble FGFs Based on their relative expression and solubility properties, the FGFs were split into three different groups: FGFs that expressed well as soluble proteins (Group 1: FGF1, FGF2 and FGF10), FGFs that expressed at a low level (Group 2: FGF3 and FGF7), and FGFs that were insoluble when expressed in E. coli (Group 3: FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22)
Summary
Of the 18 receptor-binding fibroblast growth factors (FGF), 15 bind a heparan sulfate co-receptor and are classed as growth factors and morphogens These are grouped into 5 subfamilies based on their protein sequence similarity (Itoh, 2007; Ornitz, 2000), and they regulate a myriad of processes in development, homeostasis and in some diseases (Beenken & Mohammadi, 2009; Turner & Grose, 2010). While fluorescent proteins remain a mainstay of genetic labelling, they have limitations These have been overcome, for example, by non-covalent tagging of proteins on hexahistidine sequences with Tris-Ni2+ nitriloacetic acid (Huang et al, 2009; Lata et al, 2005; Tinazli et al, 2005), which has allowed diverse labelling strategies, ranging from fluorescent dyes (Uchinomiya et al, 2009) and quantum dots (Roullier et al, 2009; Susumu et al, 2010) to gold nanoparticles (Duchesne et al, 2008). Non-covalent coupling is reversible and exchange may occur in this instance with histidine-rich patches on endogenous proteins
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