Tandem Repeat Array Construction Using CTPRa

The tandem repeat arrays of CTPRa or CTPR10D (a single CTPR with the ‘10D’ loop sequence) were built by concatemerization of individual CTPRa or CTPR10D using BamHI and BglII sites as previously described^{10 (link),29 (link)}. Briefly, a single consensus tetratricopeptide repeat (CTPRa1) was purchased as a short double-stranded DNA fragment and inserted into the T7-regulated expression vector pRSET B between the BamHI and HindIII restriction sites (ThermoFisher Scientific). The CTPRa1 fragment was then PCR-amplified using T7 promoter primers. The CTPRa1 PCR product and CTPRa1 pRSET B vector were then digested with BamHI/HindIII and BglII/HindIII restriction enzymes, respectively. The result is two concatamerized CTPRa1 genes froming a CTPRa2 (i.e. two-repeat array). The concatamerization of BamHI and BglII results in an Arg and a Ser after the highly conserved Pro31 of the CTPR sequence. As a result, the CTPRa2 contains the well-studied DPRS loop^{30 (link)}. This process can be repeated to generate CTPRa proteins of different lengths. Addition of the CTPR10D module generates the CTPRalt constructs. The sequences are given in Table S1.