Xhoi

An N-terminal 6x-histidine (NTH) tagged construct of ShGH76 was designed for Ni-NTA (nitrilotriacetic acid) based immobilized metal-ion affinity chromatography purification (IMAC, see below). The synthetic gene was cloned into the pET28a(+) vector using the NheI and XhoI restriction sites (GenScript pvt Ltd., Piscataway, NJ, USA). The WT plasmid was used as template to engineer ShGH76 mutants by site-directed mutagenesis [50 (link)]. Six mutants were constructed (D136A, D137A, D136A-D137A, D136E, D137E and D136E-D137E). Similarly, NTH tagged constructs of six GH92s were cloned into the pET28a(+) vector using combinations of the NdeI/NheI and XhoI/NotI restriction sites. The primers used to perform mutagenesis and GH92 cloning are listed in Supplementary Table S4. All clones in this study were verified by DNA sequencing.

The construct corresponding
to the TTR amino acid sequence plasmid pcDNA3.1+/C-(K) DYK with Accession
No. NM_000371 was purchased from GenScript, USA. The TTR gene was
cloned as per the protocol described by Ali et al.^{48 (link)} The plasmid pcDNA3.1+/C-(K) was amplified using PCR with
this primer set: the primers 5′-ATATATAAGCTTATGGCTTCTCATCGTCTG-3′
tagged with a 5′-HindIII cleavage site and
5′-ATAT ATCT CGA GT CATTCCT TGGGATTGG-3′ tagged with
a 5′ XhoI cleavage site. A construct corresponding
to the mature protein without the predicted signal sequence was amplified.
The PCR-amplified product was double-digested with HindIII and XhoI restriction enzymes. The pET28b vector
(GenScript) was also double-digested with HindIII
and XhoI restriction enzymes. The digested product
(insert and backbone) was ligated using the T4 DNA ligase. The ligated
vector was used to transform the Escherichia coli DH5α strain, which was plated onto Luria–Bertani (LB)
agar plates containing 50 μg/mL of kanamycin (Km). The subsequent
transformants were collected for plasmid preparation using Gene Jet
Minipreps (Thermo Scientific). The plasmids were digested with HindIII and XhoI (New England Biolabs)
to confirm the positive clones. The constructs were sequenced using
Eurofins.

The SARS-CoV-2 WT Mpro gene (UniProtKB P0DTD1; nsp5 sequence) was codon optimized for E. coli and cloned into a SUMOstar vector (LifeSensors Inc.; Malvern, PA, USA) as a C-terminal fusion to an N-terminal small ubiquitin-related modifier (SUMO) tag using BsaI and XhoI (GenScript; Piscataway, NJ, USA). This results in a construct that, upon SUMO cleavage, generates a protein with the native N-terminus. The H163A mutant was generated in the background of this WT construct (GenScript).

Synthetic DNA encoding for the 13 kDa N-terminal human DARPP-32 fragment, DARPP-32_1-122 was subcloned into pET-28a(+) (Novagen) using NdeI and XhoI restriction sites (Genscript). The resulting plasmid, pET-28trDARPP-32 confers kanamycin resistance and expresses N-terminally 6XHis-tagged DARPP-32_1-122 from the T7 lac promoter.

HLA-C*05:01 and TCR proteins were produced largely as described (Clements et al., 2002 (link); Garboczi et al., 1992 (link); Sim et al., 2020 (link); Tikhonova et al., 2012 (link)). DNA encoding residues 1–278 of HLA-C*05:01, 1–99 of β₂M and TCR alpha (1–208) and beta (1–244) chains were synthesized and cloned into the bacterial expression vector pET30a via NdeI and XhoI (GenScript). Proteins were expressed as inclusion bodies in BL21 (DE3) cells (Novagen, USA) and dissolved in 8 M urea, 0.1 M Tris pH 8. Proteins were refolded by rapid dilution in 0.5 M L-arginine, 0.1 M Tris pH 8, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione. Proteins were purified by ion-exchange chromatography (Q column, GE Healthcare, USA) followed by size-exclusion chromatography with a Superdex 200 column (GE Healthcare). HLA-C*05:01-G12D-A18L-9mer and TCR9a were concentrated to 10 mg/ml and crystals grown under the same conditions as TCR9a/d with HLA-C*08:02-G12D-9mer (Sim et al., 2020 (link)) and were 22% PEG 3350, 0.1 M MOPS pH 7.1, and 0.25 M MgSO₄.