Pet 22b plasmid

pHN1+ plasmids encoding the mature human beta 2-microglobulin protein (β2m hereafter) or the ER luminal domains of HLA A*02:01 protein with C-terminal BirA motif were obtained from Prof P Moss (University of Birmingham, UK). pGM-T7 plasmids encoding the ER luminal domains of wild-type HLA B*08:01 or the T134K point mutant, each with C-terminal fos leucine zipper sequences, were provided by Prof M Bouvier (Chen and Bouvier, 2007 (link)). The sequence encoding HLA-B*08:01fos or the T134K point mutant were excised by restriction enzyme digestion and sub-cloned into pET22b plasmid (Novagen, Merck Millipore, Germany). DNA encoding the ER luminal domains of HLA A*02:01 with C-terminal Fos leucine zipper sequence was created by PCR: nucleotides encoding the ER luminal domains of A*02:01 were amplified using primers 5'-GATATACCATGGGCTCTCACTCC-3' and 5'-CGGAACCTCCCTCCCATC-3’ and pHN1 A*02:01 DNA; while nucleotides encoding the Fos leucine zipper were amplified from pET22b HLA B*08:01fos using primers 5'- GGAGGTTCCGGCGGTC-3' and 5'-CGCAAGCTTTTAATGGGCGG-3'. The purified products from both PCR reactions were used in a third PCR reaction to create A*02:01fos using primers 5'-GATATACCATGGGCTCTCACTCC-3' and 5'-CGCAAGCTTTTAATGGGCGG-3'.
Following agarose gel electrophoresis and digestion of the purified product with restriction enzymes the sequence encoding HLA-A*02:01fos was cloned into pET22b.

A construct expressing C-terminally FLAG-tagged MOA under the control of the CaMV 35 S promoter was generated using the Gateway R Cloning Technology (Thermo Fisher Scientific, USA). The open reading frame of MOA was PCR-amplified using gene-specific primers (MOA-Fw: 5′-CACCATGTCTCTGCGACGCGG-3′, and MOA-Rev: 5′-GTAGAAGGCCATGTAGCTGTC-3′) from pET-22b(+) plasmid (Merck Millipore Novagen, USA). The PCR product was introduced into a pENTR vector (pENTR™/D-TOPO™ Cloning Kit, Thermo Fisher Scientific, USA) and the reaction products were transformed into chemically competent TOP10 E. coli cells. Positive colonies were detected by colony PCR (Biometra, Germany). The DNA sequence was confirmed by sequencing (Eurofins Genomics, Germany). The obtained entry plasmids were recombined into the binary Gateway expression vector pB2GW7 [47 ], utilizing LR reaction (Gateway™ LR Clonase™ II Enzyme mix, Thermo Fisher Scientific, USA). The colony PCR-confirmed binary expression plasmid (35 S::MOA-3xFLAG) was transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method [48 (link)].

The hcp1 gene from B. pseudomallei was cloned in frame with the C-terminus His-tag of the pET-22b plasmid (Merck KGaA) and transformed into BL21 (DE3). Recombinant Hcp was purified using B-PER 6xHis Fusion Protein Purification Kit. Site-specific mutations in Hcp1 were introduced by overlapping PCR as previously described38 (link) and verified by DNA sequencing.