Cloning and expression of GRP-hFGF5 fusion protein

The DNA fragment coding residues 1–119 of Bombyx mori ß-1,3-glucan recognition protein (GRP) and the cleavage site of HRV 3C protease (3C) was amplified with polymerase chain reaction using KOD plus neo DNA polymerase (TOYOBO CO., LTD. Osaka, Japan) with the primers 5′-CATGCCATGGAGTACGAGGCACCACCGGC-3′ and 5′-GGAATTCCATATGCGGGCCCTGAAACAGCACTTCCAGAAATTCTACTCCTGGTGTTATTTCAGAG-3′ from pET-GRP-3C-His as a template^{41 (link)}. The DNA fragment coding hFGF5 residues 21–242 [hFGF5 (21–242)] (GenBank id: NM_004464.3) was amplified with the primers 5′-CACCCATATGCACGGGGAGAAGCGTCTCG-3′ and 5′-GCCCTCGAGAGGGCTAGGTGGCTTTTTCTTTTCAG-3′ from Human Universal QUICK-Clone cDNA II (Takara Bio USA, Inc., CA, USA) as a template. DNA fragments of GRP-3C and hFGF5 (21–242) were cloned into pET-21d ( +) (Merck KGaA, Darmstadt, Germany) to give pET-GRP-3C-hFGF5 (21–242)-His.