Pet28a expression vector

Sodium dodecyl sulfate (SDS), alginate (A7003), phenylmethylsulfonylfluoride (PMSF), imidazole, and dithiothreitol (DTT) were obtained from Sigma Aldrich (St. Louis, MO), whereas cloned pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). DpnI endonuclease, protein A-horseradish peroxidase (HRP), polyvinylidene fluoride, and pET28A(+) expression vector were obtained from New England BioLabs (Ipswich, MA), BD Transduction Laboratories (BD Biosciences, Franklin Lakes, NJ), EMD Millipore (Billerica, MA), and Invitrogen (Waltham, MA), respectively. The QIAprep Spin Plasmid Miniprep Kit and Ni-nitrilotriacetic acid (NTA) agarose beads were purchased from Qiagen (Venlo, Netherlands). The ECL Plus Western blotting detection system RPN 2132 was obtained from Amersham Life Science (GE Healthcare, Cleveland, OH). The oligonucleotide primers were synthesized by Bioneer (Daejeon, Korea) and the analysis facility of Chosun University. Acetylated alginate was purified from Pseudomonas as previously described, using an alginate-overproducer, P. alkylphenolia E1 (pAlgG). Purified acetylated alginate was converted to a Na-form by treating with 50 mM ethylene diamine tetraacetic acid (EDTA), and dialysis using 0.5 M NaCl and double distilled water [41 ].

The anti-mKRAS G12V-34 scFv and enhanced green fluorescent protein (eGFP) were amplified separately and were purified using the gel extraction kit (iNtRON, Korea). The PCR products were assembled via SOE-PCR and double digested with XbaI and HindIII (NEB, Hitchin, UK). The digested PCR products were ligated into pET-28a expression vector (a molar vector to insert ratio of 1:3), using electroligase (NEB, Hitchin, UK). The ligation products were electroporated into E.coli BL21(DE3) at 1.8 kV, 5 ms. One milliliter of pre-warmed SOC media was added to the electroporated cells, which were then incubated at 37 °C for 1 h with shaking at 150 rpm. The recovered cells were plated out into LB-kan plates and incubated at 37 °C overnight. Positive clones were isolated with iNtRON Biotechnology DNA-spin^™ plasmid DNA purification kit according to the manufacturer’s protocol. The scFv-eGFP DNA region was sequenced using pET22b sequencing forward primer 5′-TAATACGACTCACTATAGGG-3′. Positive scFv-eGFP clones were expressed in E. coli BL21(DE3) in the presence of 1 mM IPTG for 4 h at 37 °C with shaking at 200 rpm. The cells were harvested by centrifugation at 7,000×g for 10 min and resuspended in 20 mM Tris-CI buffer (pH 8.0) with the addition of 1 mM phenylmethylsulfonyl fluoride (PMSF) prior to sonication at 130 watt and 20 kHz for a total of 10 min on ice.