The pIGF5 plasmid DNA was isolated from E. coli using the commercial Quantum Prep plasmid Kit (Bio-Rad) following the vendor’s instructions to obtain high-purity DNA. Next, this plasmid was linearized by using the Sac I restriction enzyme (Invitrogen), and 8 μg of linearized plasmid DNA was used to transform P. pastoris SuperMan5 electrocompetent cells. The transformation was carried out via electroporation in a MicroPulser Electroporator (Bio-Rad), according to the pre-established parameters for P. pastoris (1500 V, 200 Ω, and 25 mF; [37 ]. After 96 h of incubation at 30 °C in the presence of the 200 μg/mL zeocin selective antibiotic, zeocin-resistant clones were isolated for analysis of the integration of the hLf gene by PCR.
Sac 1 restriction enzyme
Manufactured by Thermo Fisher Scientific
Sourced in Brazil
Sac I is a type II restriction enzyme that recognizes and cleaves the palindromic DNA sequence 'GAGCTC'. It is commonly used in molecular biology and genetic engineering applications for the manipulation and analysis of DNA sequences.
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Lab products found in correlation
2 protocols using sac 1 restriction enzyme
1
Isolation and Transformation of pIGF5 Plasmid in P. pastoris
2
Optimized Expression of BoHV-1/5 gE Protein
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Following the alignment of six gE proteins (GenBank accession numbers: NP_045372.1, AAA67214.1, ADE08265.1, AAF71143.1, YP_003662535.1, AAF34744.1) we identified a polypeptide that included highly conserved regions in both BoHV-1 and BoHV-5. This was encoded by a 1682 bp sequence from BoHV-1 and was named BoHV-1/5 gE. The codon usage of the gE sequence was optimized for P. pastoris (based on the codon usage database at http://www.kazusa.or.jp/codon). Restriction enzyme sites for EcoRI and KpnI, at the 5'-and 3'-termini, respectively, were included in the gE sequence for subsequent cloning into the pPICZaB expression vector (Invitrogen, Brazil). The presence of the insert was determined by restriction enzyme digestion with EcoRI and KpnI. The pPICZaB/BoHV-1/5 gE plasmid was propagated in E. coli DH5a, purified (Purelink Quick Mini 50 Kit, Invitrogen, Brazil) and linearized with SacI restriction enzyme (Invitrogen, Brazil). Linear plasmid DNA was purified by phenol-chloroform extraction and DNA precipitation as previously described [44] . P. pastoris competent cells were transformed with 10 mg of linear plasmid DNA by electroporation (25 mF, 200 U, 2 kV).
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