Hbl proteins (rHbl B and L2 with N-terminal and rHbl L1 with C-terminal strep tag) were overexpressed in E. coli BL21 (DE3) and purified as described earlier [26 (link)]. In the present study, a system for separation of the tag was established. The genes hblC (L2), hblD (L1) and hblA (B) were amplified by PCR using pASK-IBA5+hblC, pASK-IBA3+hblD and pASK-IBA5 + hblA [26 (link)] as templates, as well as the primer pairs L1_7+_fw (ATATCCGCGGTGCACAAGAAACGACCG) and L1_7+_rev (ATATGTCGACCTACTCCTGTTTAAAAGCAATATC), L2_7+_fw (ATATCCGCGGTCAAGCAGAAACTCAACAAGA) and L2_7+_rev (ATATGTCGACTCAAAATTTATACACTTGTTCTTC), and B_7+_fw (ATATCCGCGGTGCAAGTGAAATTGAACAAAC) and B_7+_rev (ATATGTCGACCTATTTTTGTGGAGTAACAGTTTC). Using the enzymes SacII and SalI (New England Biolabs, Frankfurt, Germany; restriction sites underlined in the primer sequences), the three genes lacking the sequences for the N-terminal signal peptides for secretion [64 (link),65 (link)] were cloned into pASK-IBA7+ (IBA Lifesciences, Göttingen, Germany). The constructs were sequenced using the primers pASK-IBA-seq-fw (CACTCCCTATCAGTGATAG) and pASK-IBA-seq-rev (GCACAAT GTGCGCCAT). Proteins were again overexpressed in E. coli BL21 (DE3) and purified via their N-terminal strep tag as described earlier [26 (link)].
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