Alicator ligation independent cloning and expression system

Primer design, amplification, cloning and expression of lngB and lng genes in the pLATE31 plasmid were conducted according to the aLICator Ligation Independent Cloning and Expression System (Thermo Scientific, CA, USA). pLATE31 and the recombinant expression plasmid pLATE31-lngB and pLATE31-lngC were transformed into E. coli BL21 (DE3). For the expression and detection of LngB and LngC, E. coli was cultured in LB medium at 37°C, and induced with 1 mM IPTG for 5 h. LngB and LngC His-Tag recombinant proteins were purified from 200 ml of induced cultures media. The cultures were centrifuged at 3500 × g, and the pellets were resuspended in 20 ml of pH 8.2 lysis buffer (8M Urea, 100 mM NaH₂PO₄, 10 mM Tris-HCl, and 10 mM imidazole) and sonicated for 30 min at 40 kHz, using 10 cycles of 1 min pulse and 2 min of cooldown. The sonicated samples were centrifuged at 3500 × g, and the His-tag recombinant proteins from the clear supernatants were captured in an affinity chromatography column of Ni-NTA Agarose (Qiagen, Hilden, Germany), washed with 60 ml of lysis buffer (pH 6.5), and eluted with 10 ml of lysis buffer (pH 4.0). The eluted fractions containing the recombinant proteins were refolded by dialysis and maintained in phosphate-buffered saline (PBS) pH 7.0. Anti His-Tag monoclonal antibodies were used to detect the expression of recombinant proteins by immunoblotting.

MlrB and mlrC ORFs were amplified using Sphingomonas sp. ACM-3692 genomic DNA as a template. After amplification with mlrBF and mlrBR primers (Table 3), mlrB was inserted into pLATE31 vector using an aLICator Ligation Independent Cloning and Expression System (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. The resulting plasmid (pLATE31mlrB) encodes full-length MlrB (541 amino acid residues) with a six-histidine tag added to C-terminus. The amplified mlrC fragment was inserted into a pTZ57R/T cloning vector, cut out using NdeI and NotI restriction enzymes, and inserted into the expression vector pET21a. The resulting plasmid pET21amlrC encodes full length MlrC (507 amino acids) with a His tag on its C-terminal. Particular muteins were prepared based on the initial constructs pLATE31mlrB and pET21amlrC by site-directed mutagenesis method. Codons selected for the mutagenesis were replaced with codon for alanine introduced with primers (Table 4). The PCR was carried out with a primer pair introducing a mutation (Table 3) and Phusion polymerase.

The recombinant plasmid for C-terminal His-tagged R27-encoded E protein overexpression was obtained following the instructions of ALICator Ligation Independent Cloning and Expression System (Thermo Scientific) using primers RepECtermFor and RepECtermRev (Supplementary Table S1). Recombinant clones were sequenced before overexpression.
Transformed BL21(DE3) cells were grown at 37°C in LB until the cultures reached an OD_600nm of 0.5–0.6. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was subsequently added to a final concentration of 1 mM. Upon 3 h incubation at 37°C, cells were centrifuged and pellets were frozen, resuspended in lysis buffer (50 mM NaH₂PO₄ pH 8, 1 M KCl, 10 mM imidazole suplemented with 1 mg ml^-1) of lysozyme and a protease inhibitor cocktail (cOmplete ULTRA Tablets Mini, EDTA-free, EASYpack Roche) and disrupted by sonication on ice. The lysate was centrifuged at 20,000 × g for 30 min at 4°C, and the supernatant was then treated with Ni-NTA (Ni²⁺-nitrilotriacetate)–agarose (Qiagen). The resin was washed extensively with the same buffer, and the protein was eluted using 200 mM imidazole as described previously (Nieto et al., 2000 (link)).