T4 dna ligase enzyme

The gene-related amplicons were cloned into the pAE expression vector [27 (link)], according to the restriction sites established for each ORF (Open Reading Frame), as indicated in Table 1. For this purpose, each amplification product and the vector were digested with the respective restriction enzymes. Then, both were ligated at room temperature in a solution containing 1 μl of the T4 DNA ligase enzyme (Invitrogen), 0.5 μl of the vector, 2 μl of buffer, and 15.5 μl of MiliQ H₂O. The products of the constructs were transformed into E. coli TOP 10 electrocompetent cells and seeded on plates containing LB medium with ampicillin (100 μg/ml) and incubated for 16 h at 37°C. The obtained colonies were submitted to screening by lysis with phenol-chloroform (v/v) and analyzed in 1% agarose gel. The selected clones were cultured in LB broth with ampicillin (37°C/140 rpm for 12–16 h) before extracting the plasmid DNA using the Plasmid Miniprep Spin kit (GE Healthcare Life Sciences). Confirmation of the presence of each gene in the recombinant clones was carried out by digestion with the restriction enzymes used in cloning the pAE vector.

The human fusion ORF cDNA sequences for NCOA4 were amplified from MCF7 (ATCC® HTB-22^™) cell line and ΔRET was amplified from pDONR223-RET, a gift from William Hahn & David Root (Addgene plasmid #23906) using recombinant Taq DNA polymerase enzyme (Life Technologies) with fusion-specific primers (Supplementary Table 3). Gel-extracted PCR products for NCOA4-RET were ligated using T4 DNA ligase enzyme (Invitrogen). The predicted ORFs for NCOA4-RET, ΔRET and full-length, wild-type RET (RET^amp) were cloned into pLenti6.3/V5–DEST and pEF-DEST51 vectors (Invitrogen) that include a C-terminal V5-tag. Constitutively kinase active (corresponding to M918T) and kinase inactive (corresponding to K758M) full-length variants were generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent). All constructs were verified by DNA sequencing.

Cloning, recombinant protein production and protein purification were performed as previously described [13 ]. The pAE vector [14 (link)] and the amplified cp40 gene were digested using BamHI and EcoRI restriction enzymes (Fermentas, USA). After that, the cp40 gene was inserted into the pAE vector using the T4 DNA ligase enzyme (Fermentas, USA), and electrocompetent TOP 10 E. coli cells were transformed by electroporation. The transformed bacteria were cultivated in Luria-Bertani (LB) broth with 100 μg/mL ampicillin for 16 h, and the recombinant plasmid was purified using a MiniPrep Kit (Qiagen, USA). The pAE/CP40 recombinant plasmid was transformed by thermal shock in E. coli BL21, and the recombinant bacteria were cultivated in LB broth containing 1 mM IPTG for 3 h at 37°C in an orbital shaker.
For recombinant protein purification, the bacteria resulting from a large-scale culture (500 mL) were pelleted by centrifugation and resuspended in a washing buffer (200 mM NaH₂PO₄; 500 mM NaCl, 5 mM Imidazole; 8 M Urea pH 8.0) with 100 mg/mL lysozyme, sonicated five times for fifteen seconds (20 KHz) and maintained under agitation at 4°C for 16 h. The recombinant protein was purified using the HisTrap^TM HP (GE Healthcare, USA) affinity chromatography column followed by dialysis in a cellulose tube-based system (Sigma, USA).