Centricon microconcentrators

To prepare an expression construct to produce the ABP-Cn-AFP fusion protein, the 5′-forward primer encoding the aluminum-binding peptide (VPSSGPQDTRTT; shown in bold in Table 1) was used. The 5′- and 3′-primers included the XhoI and SalI restriction sites, respectively (underlined in Table 1). The open reading frame (ORF) encoding the active form of Cn-AFP (without a signal peptide) was amplified by PCR using the genomic DNA of C. neogracile, and was used to produce Cn-AFP_G124Y via site-directed mutagenesis, as described in our previous report16 (link). Cn-AFP genes were amplified by PCR, digested with XhoI and SalI, and ligated into the pColdI expression vector (Takara, Japan). The expression vectors were transformed into E. coli BL21 (DE3), and the pColdI expression method (Takara, Japan) was used to induce protein production. Cells were disrupted by sonication and soluble recombinant proteins were purified by His-tag affinity chromatography (Qiagen, U.S.A)16 (link). Purified proteins were concentrated by Centricon microconcentrators (Millipore, U.S.A.) and the protein concentration was measured using the Bradford reagent (BioRad, U.S.A.).