Ncoi and hindiii

V_H and V_L were cloned into pET28 A (Novagen, Darmstadt,Germany) with NcoI and HindIII (NEB, Hitchin, UK) and expressed in E. coli BL21 star (Invitrogen, Carlsbad, USA). The transformed cells were grown in LB medium containing kanamycin at 37 °C until an OD₆₀₀ of 0.6-0.8 was reached. The expression was induced by the addition of 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). After 12 h, cells were harvested, and preparation of inclusion bodies was carried out as described previously^{23 (link)}. The purification was performed according to the procedure described for the V_L domain^{24 (link)}. Protein purity was checked by SDS-PAGE (Fig. S7).
Single point mutations were introduced by a quick change PCR approach using the QuikChange® Site-Directed Mutagenesis Kit (Agilent Technologies Inc., Santa Clara, USA) according to the manufacturer’s recommendations. Primers were ordered from MWG Operon (Ebersberg, Gemany). Intact protein was verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Strains used for the propagation of the plasmid DNA—E. coli DH5α, DH10BAC, for the expression of recombinant proteins cells—E. coli BL21 (DE3), insect cell line Sf21 and the cloning vectors pRSET-B, pFASTBAC-HTB, pGEM-3Z and pGEM–T Easy were procured from Invitrogen, USA. The restriction enzymes KpnI, NcoI and HindIII, Calf-Intestinal Alkaline Phosphatase, Pfu and Taq polymerases were obtained from New England Biolabs, USA. ATP and T7 RNA polymerase were purchased from Fermentas, USA. Nickel-NTA beads, DEAE-cellulose columns and plasmid midi-prep kit were procured from Qiagen, USA. PD-10 columns were purchased from GE Healthcare Life Sciences, USA. Trizol, DAB (3,3′-diaminobenzidine), Lysozyme, anti-His monoclonal antibody, plasmid miniprep kit and protease inhibitor cocktail were purchased from Sigma Chemicals, USA. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was acquired from GIBCO-URL, USA. An antibody was raised in rabbit earlier against recombinant PPRV L protein domain 3 (1717–2183 a.a) expressed in E. coli [3 (link)]. γ-P³²-ATP and α-P³²-ATP were purchased from BRIT, Mumbai, India. The oligonucleotides were supplied by Sigma Chemical Co., India and were used to generate in vitro transcribed RNA substrate for RTPase assays.

The coding sequences of β-lactamase and DeepBL candidate genes were amplified from FK688 gDNA using Fusion High-Fidelity DNA polymerase (New England BioLabs) with the oligonucleotide primers listed in Table 6. The PCR products and the anhydrotetracycline (ATc)-inducible expression vector pJP-CmR were digested with restriction enzymes using either NcoI and HindIII, or EcoRI and HindIII (New England BioLabs) and ligated to create the plasmids listed in Table 5. Plasmids were verified by sequencing, transformed into E. coli BW25113 or K. quasipneumoniae FK688, and selected with chloramphenicol. Target gene expression was induced with 35 ng/mL ATc. The parental plasmid pJP-CmR was used as the control in all experiments.