Puc19

The A47L homology donor was synthesized and cloned into pUC19 (Genscript). The C1L homology donor was generated by PCR using VC2 (wild-type vaccinia virus strain Copenhagen) DNA as a template and sequential restriction digest-based cloning for the 3′ and 5′ homology arms. See annotated primer table (Table S3) for details.

The Elastin-like Peptide (ELP) used as a part of the fusion protein is L10FLAG, which is denoted by the sequence^{27 (link)}[(VPGVG)₂(VPGLG)(VPGVG)₂]₁₀DYKDDDDK. PMP-D2 is denoted by the amino acid sequence EEKCTPGQVKQQDCNTCTCTPTGVWGCTLMGCQPA. APP-IP is denoted by the amino sequence ISYGNDALMP. The nucleic acid sequences for the three amino acid sequences used to create PMP-D2٠L10FLAG٠APP-IP were obtained in plasmid form in pUC19 or pUC57 from GenScript^®. The gene for PMPD2-L10FLAG-APP-IP was constructed by cutting PMPD2 from a pUC57 plasmid using PflMI and BglI restriction enzymes^{19 (link)}. L10FLAG pUC19 was linearized using PflMI restriction enzyme^{19 (link)}. A ligation was performed between the PMP-D2 fragment and the linearized L10FLAG pUC19 to form PMPD2-L10FLAG pUC19 plasmid. Then PMPD2-L10FLAG was cut out of the pUC19 plasmid using PflMI and BglI restriction enzymes^{19 (link)}, while APP-IP pUC57 plasmid was linearized using PflMI. A ligation was performed between the PMPD2L10FLAG fragment and the linearized APP-IP pUC57 to form PMPD2-L10FLAG-APP-IP pUC57 plasmid. Using the same techniques PMPD2-L10FLAG and APP-IP-L10FLAG was created to use as comparable inhibitors of NE and MMP-2 respectively. Figure 1 displays the cloning scheme for the construction of the plasmid containing PMPD2-L10FLAG-APP-IP.