Saci restriction enzyme

Escherichia coli TOP10F' strain was used for the amplification of the expression construct pPICZαC/FoFaecMut (Eurofins Genomics, Luxembourg) and the transformants were selected on Low Salt Luria-Bertani medium (1% tryptone; 0.5% yeast extract; 0.5% NaCl pH 7.5) by Zeocin™ resistance (25 μg mL⁻¹). The resistant transformants were grown overnight at 37°C under shaking and plasmid DNA was isolated by the Plasmid DNA Extraction Mini Prep Kit (Fisher Molecular Biology, Rome, Italy). The recombinant plasmid pPICZαC/FoFaecMut was linearized with SacI restriction enzyme (NEB, Ipswich, MA, USA) to transform Pichia pastoris X-33 (Invitrogen, Carlsbad, CA, USA). The transformation of yeast was performed with 5 μg pure recombinant vector by Electroporation protocol according to the EasySelect™ Pichia Expression Kit (Invitrogen, Carlsbad, CA, USA). P. pastoris transformants were selected on YPDS agar (1% w/v yeast extract; 2% w/v peptone; 2% w/v dextrose; 1 M sorbitol; 2% w/v agar) containing Zeocin™ at final concentration of 100 μg mL⁻¹at 28°C. Thirty selected transformants were grown in BMGY and BMMY (1% w/v yeast extract; 2% w/v peptone; 100 mM potassium phosphate, pH 6.0; 1.34% w/v YNB; 4 × 10⁻⁵% w/v biotin; 1% v/v glycerol or 0.5% v/v methanol) at 28°C.

Cloning was performed in three steps. First, the pmCherry-C1 vector was cleaved with SacI restriction enzyme (New England Biolabs), and the cohesive ends were blunted with T4 DNA polymerase (New England Biolabs) and ligated with T4 DNA ligase (New England Biolabs). Second, the resulting vector was cleaved with HindIII and XbaI restriction enzymes (New England Bioloabs). The same pair of restriction enzymes was used to generate the insert from the pEGFP-N1 plasmid, which contains the EGFP open reading frame. These two fragments were ligated. Third, the resulting mammalian expression vector containing the mCherry-polylinker-EGFP open reading frame was cleaved with HindIII and KpnI restriction enzymes (New England Biolabs). The HIV-1 protease precursor was obtained by PCR amplification from the pNL4-3 plasmid using the primer pair 5′-TTTAAGCTTTTTTTTAGGGAAGATCTGGCC-3′ and 5′-TTTTGGTACCGTCTTTAATTTTACTGGTACAGTC-3′. The PCR product was cleaved with HindIII and KpnI restriction endonucleases and ligated into the vector.

Genomic DNA was extracted from peripheral blood cells according to a modified Miller technique. Genotyping was performed using polymerase chain reaction followed by a restriction fragment length polymorphism (PCR-RFLP) technique for the PTPN22 −1123G>C and +1858C>T polymorphisms. PCR products were digested for one hour at 37°C with appropriate restriction enzymes and then resolved on a 6% polyacrylamide gel.
For the −1123G>C polymorphism a 205 bp fragment was amplified (following our previously reported method) [26 (link)] and digested with Sac I restriction enzyme (New England Biolabs, Ipswich, MA, USA). The polymorphic allele −1123C was cleaved in two fragments of 183 bp and 22 bp, respectively, while the −1123G allele remained intact. For the +1858C>T SNP we used previously reported conditions [14 (link)]. Amplified products of 412 bp were digested with Xcm I restriction enzyme (New England Biolabs, Ipswich, MA, USA); only the polymorphic +1858T allele was cleaved into two fragments of 246 bp and 166 bp. Results were confirmed by automatized sequencing of one random sample of each genotype (Applied Biosystems, USA).

Both mouse and human α211 constructs were linearized by digesting with SacI restriction enzyme (New England Biolabs) and transformed into KM71H of P. pastoris (Invitrogen) by electroporation. The transformants were plated on YPDS plates, which contained 100 µg mL⁻¹ Zeocin. Plates were incubated at 30°C for 3–5 days until colonies formed, and several colonies were restreaked on fresh YPDS plates. Pre-inoculation was made by seeding a single colony in 30 mL BMGY medium. The culture was incubated at 30°C with shaking overnight. About 5–7 mL of this culture was used to inoculate 500 mL of BMGY in a 2 L baffled flask (total 2 L of culture). The inoculated culture was incubated at 30°C with shaking to OD₆₀₀ value of 6. Cells were harvested by centrifugation at 3000×g for 15 min at room temperature. The supernatant was discarded, and cell pellets were resuspended in 400 mL of BMMY medium for induction. The resuspended culture was divided between two 2 L baffled flasks (200 mL each) and incubated at 20°C with shaking for 72 hr. 100% methanol was added every 24 hr to a final concentration of 0.5% (v/v) to induce protein expression. After 72 hr of induction, cells were harvested by centrifuging at 6000×g for 20 min at room temperature. Protein purification proceeded with the supernatant as the protein was secreted.