Expression vector design

The expression vectors pET32b-1XHis-scFv, pET32b-3XHis-scFv, and pET32b-5XHis-scFv were constructed for Thy1-scFv expression. A ligation substrate featuring Thy1-scFv protein was amplified by PCR using a forward primer with NcoI restriction enzyme site and a reverse primer with XhoI restriction enzyme site. The amplified fragment was digested with NcoI and XhoI and ligated into pET-32b(+) prokaryotic expression vector digested with respective restriction enzymes to construct pET32b-1XThy1-scFv with a single inherent hexa-histidine-tag located between the Trx- and S-tags. To introduce more hexa-histidine-tags to construct pET32b-3XHis-scFv and pET32b-5XHis-scFv vectors, we inserted annealed forward and reverse primers coding for 2 and 4 additional hexa-histidine-tags (i.e., 3XHis and 5XHis total) with BglII restriction enzyme site on both the sides as overhangs with 5′-phosphate group. After ligation into pET32b-1XThy1-scFv vector previously digested with BglII restriction enzyme and dephosphorylated using Calf intestine alkaline phosphatase, we generated two additional vectors with 3X and 5X hexa-histidine-tags. All three plasmids contain: a T7 promotor; two fusion partners Trx- and S-tags for enhancing protein folding and solubility; 1, 3 or 5 hexa-histidine tag(s) for purification by immobilized metal affinity chromatography (IMAC); a DDDDK sequence on the N terminus of Thy1-scFv for tag removal using EK cleavage; and the Thy1-scFv gene. Each histidine tag was separated from each other by a few amino acid residues to increase flexible folding. The sequence confirmed, vectors were transformed into SHuffle T7 E. coli cells (New England Biolabs, Ipswich, MA) for recombinant protein expression. Oligonucleotides and recombinant protein sequences used in this study for constructing the vectors are listed in Supporting Information (Supplementary Table ^S1).