Cloning and Purification of Sema5A Constructs

The constructs used in this study were cloned into pHLsec^{44 (link)} or pcDNA 1.1 variant vectors with relevant tags for expression in cell lines. A construct of human Semaphorin-5A (Sema5A) (UniProtKB: Q13591), Sema5A_sema-TSR1-7, (residues 23E-944S) was cloned into the pHLsec vector in-frame with a C-terminal hexahistidine (His₆) tag. Constructs of Sema5A_sema-TSR2 (residues 23E-651P) and Sema5A_TSR3-4 (r. 652P-765T) were cloned into the pHLsec vector in-frame with a C-terminal 3C-Avi-His₆ tag. For protein purification, we used pHLsec vectors which also code for a C-terminal His₆-tag or a C-terminal 3C-Avi-His₆-tag, for BLI and GAGOme assay we used a C-terminal 3C-Avi-His₆-tag. A construct of human Sema5A Sema5A_sema-TSR1-7, (residues 23E-944S), harboring the R676C mutation was gene synthesized by Genescript and was subcloned into pHLsec vector. Primers used for cloning and mutagenesis are summarized in Supplementary Table 2.