Cloning and Expression of SUMO Variants

The previously described dicistronic plasmids pcDNA5/FRT/TO/His-S-SUMO1/IRES/HA-Ubc9 and pcDNA5/FRT/TO/His-S-SUMO3/IRES/HA-Ubc9, coding for an HA-tagged Ubc9 protein (downstream cistron) and His-S-tagged SUMO1 and SUMO3, respectively (upstream cistron)^{69 (link)}, were used as starting parental plasmids for all the expression plasmids used in this report. All clonings requiring the assembly of two different fragments of DNA were performed using PCR amplification of the desired fragments that needed to be “stitched” together, and Gibson assembly cloning^{70 (link)} using the NEB Gibson Assembly® Cloning Kit (New England BioLabs, Inc.). All clonings that required the PCR amplification of a single fragment followed by its re-circularization were performed using the NEB Quick Ligation™ Kit (New England BioLabs, Inc.). All PCR amplifications performed for cloning purposes were performed using the Q5® High-Fidelity DNA Polymerase from NEB (New England BioLabs, Inc.). The pcDNA5/FRT/TO/His-S-SUMO2/IRES/HA-Ubc9, coding for His-S-SUMO2, was produced by substituting SUMO2 for SUMO1 in the pcDNA5/FRT/TO/His-S-SUMO1/IRES/HA-Ubc9 construct. To this end, we used backbone-specific primers to amplify the backbone of the plasmid without amplifying SUMO1, and a PCR-amplified SUMO2 made using total RNA from HEK293A cells as template. The two PCR products were assembled together using Gibson assembly. To produce the SUMO1α and SUMO2α coding constructs, the parental plasmids indicated above, coding for the prototypical SUMOs, were used as templates and primers were designed to specifically delete the sequences eliminated during alternative splicing. The resulting PCR products were re-circularized using quick ligation. To produce the SUMO3α coding construct, primers were designed to amplify the full-length of the pcDNA5/FRT/TO/His-S-SUMO3/IRES/HA-Ubc9 plasmid and produce a linear product with ends located around the region where the additional sequence is introduced by alternative splicing of the transcript. The additional sequence, corresponding to the intronic extension of exon 2, was produced by using two long oligonucleotides covering the desired additional sequence and providing for two overlaps, one with the ends of the PCR-amplified linearized parental construct, and one with each other. The two primers were designed to run in anti-parallel directions, and the overlap with each other was limited to 30 bases at their 3’ ends. The hybridized long oligonucleotides were used as templates for a PCR reaction that included additional forward and reverse primers, which targeted the ends of the templates in anti-parallel direction. Such PCR reaction generated a product ready for Gibson assembly with the PCR-linearized parental plasmid. The His-S-YFP-tagged constructs were developed by PCR-amplifying the entire sequence of the parental clones using primers targeting the sequence located downstream of the His-S-tag sequence. As those sequences were shared by all the parental clones, the same set of primers were used in all of the amplifications. The coding sequence for YFP was amplified using the pEYFP plasmid (Addgene, Watertown, MA) as template. The PCR products corresponding to the linearized parental clones and the YFP coding sequence were stitched together in independent reactions (one per parental plasmid) using the Gibson assembly method. All the recombinant plasmids generated were amplified in NEB® 10-beta E. coli cells and their sequence confirmed by DNA sequencing as above. Detailed information related to the cloning methods used is available upon request.
All recombinant DNA protocols, including the use of IAV, were approved by the Institutional Biosafety Committee (IBC) at The University of Texas at El Paso (UTEP).