3.1. Construction and expression analyses of circularly permuted variants cp-hFGF7s
Conventional approaches that have attempted to produce the recombinant hFGF7 in E. coli have limitations in improving soluble expression and stability.8, 9, 11 As an alternative, we attempted to use a typical technique for inducing changes in the N- and C-terminal ends through the generation of new termini by using CP. CP, a phenomenon of rearrangement of such protein structures, can alter functional properties such as solubility and/or stability by generating new terminal regions from interior residues. Since a variant of the cytokine interleukin 4 had been successfully reported,34 the CP technique has been presumed to be applicable for producing variants with novel properties in small single-domain proteins.
Practically, plausible CP sites had been selected through the combination of prediction results from two web-based tools (CPred26 and PSIPRED29) because a deposited 3D structure (PDB: 1QQK) of hFGF7 was generated from a variant with N-terminal deletion and thus had different sequence from the wild type hFGF7 (Accession No. P21781) (Figure S1). Two high-scoring sites in a long loop (K100 to N107) and heparin binding region (K149 to K153)35, 36 were arbitrary excluded to retain structural and functional integrity (Table S2). Using the selected sequences, specific PCR was performed using the tandemly duplicated hFGF7 gene as a template and a set of primers spanning the permutable loop residues (Figure 2A). The resulting DNAs were subcloned into a redesigned vector pSCold that removed the TEE-6×His-fXa cleavage sequence from the original pCold I vector, enabling direct comparison of the expression level and solubility of cp-hFGF7 to those of the wild type hFGF7 produced without any tag at the N-terminus. As shown in Figure 2B, when the protein expression was induced with 0.2 mM IPTG at 16℃ for 36 h, the expression pattern of each clone harboring a pSCold_cp-hFGF7 variant was quite different from each other. Five CP variants were expressed distinctly and three variants were hardly detected in the soluble and/or insoluble fractions. Changes in the cultivation conditions under different media and temperatures did not significantly improve the expression properties. Intriguingly, the CP variant cp-hFGF7115-114 revealed an enhanced expression level and solubility when compared with those of the wild type hFGF7, thus it was selected to be a candidate for further analyses.