Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation

Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli. Human fibroblast growth factor 7 (hFGF7) was considered a practical drug candidate due to its cell proliferation, migration, and would healing activities. However, hFGF7 is categorized as a difficult-to-express protein in Escherichia coli and thus a method for the soluble production of a recombinant hFGF7 with promising properties is needed. A circularly permuted variant cp-hFGF7115-114 was developed without any deletion of the wild type hFGF7. This protein has proven to be more soluble and stabile than the wild type, and is comparable to or more biologically active than the wild type protein. Therefore, we expected that the cp-hFGF7115-114 variant would be a potential alternative as a drug candidate for related symptoms. In addition, the circular permutation technique was broadly applicable for the generation of functionally soluble variants of FGF family proteins. image