2.?Experimental Section2.1. Reagents and MaterialsRestriction enzymes were purchased from New England Biolabs (Beverly, MA, USA). Agarose was from Cambrex Calcitriol cost BioScience Rockland (Rockland, ME, USA). 30% (w/v) acrylamide/bis solution and protein assay were purchased from Bio-Rad (Hercules, CA, USA). HBsAg PreS2 peptide (H2N-NSTTFHQALLDPRVRGLYFPAGG-COOH) was synthesized at Peptron (Daejeon, Korea). Ni-NTA affinity kit was from Qiagen (Hilden, Germany). Other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. All oligonucleotides were synthesized at Bioneer (Daejeon, Korea).2.2. ApparatusPolymerase chain reaction (PCR) experiments were performed with a PCR Thermal Cycler (Bio-Rad) using High-Fidelity PCR System (Boehringer Mannheim, Mannheim, Germany).
DNA sequences were confirmed by automatic DNA sequencer (ABI Prism model 377, Perkin Elmer, Grove, IL, USA). Cell growth was monitored by measuring the absorbance at 600 nm (OD600; DU?650 spectrophotometer, Beckman, Fullerton, CA, USA). Cells were disrupted by using ultra-sonicator (Braun Ultrasonics, Danbury, CT, USA). SPR experiments were performed by using SPRLAB? system (K-MAC, Daejeon, Korea) and BIAcore3000 (GE Healthcare, Uppsala, Sweden). Electrochemical detection analysis was carried out using CHI 660C Electrochemical Analyzer/Workstation (CH Instruments, Austin, TX, USA).2.3. Production of 6HGBP-ScFv Fusion ProteinThe flow-chart for the construction of the fusion protein between the GBP and its single-chain antibody (ScFv) is shown in Figure S1 (Supplementary material).
The 6HGBP-ScFv fusion protein was prepared by genetically fusing the GBP and ScFv, allowing two specific interactions Cilengitide between GBP and gold substrates, and the capture of HBsAg and its ScFv, respectively. For easy purification of the fusion protein by metal affinity chromatography, the coding sequence of a six-histidine (6H) was introduced at the N-terminus of the GBP. For the cloning of the fusion gene, the DNA fragments encoding 6histidine-fused GBP (6HGBP) were obtained by PCR amplification using plasmid pTacFadLGBP-1 [16] as a template, and P1 (5��-AAAATACCATATGGGCCACCATCACCATCACCACGG-3��) and P2 (5��-TTCCCCATGGAGACGAATGGTACCGCTCGT-3��) as primers. The PCR product was digested with NdeI and NcoI, and ligated into the same sites of pET-22b(+) (Novagen, San Diego, CA, USA) to make pET-6HGBP.
For the cloning and expression of the 6HGBP-ScFv fusion gene, the DNA sequence encoding ScFv fragment was amplified by PCR using plasmid pET-ScFv-SBD [18] as a template, and P3 (5��-CAAGACCATGGGTGTCGACTGAGGAGTCTGGA-3��) and P4 (5��-TCCGCTCGAGACGTTTTATTTCCAGGTAGGT-3��) as primers. This PCR product was digested with sellekchem NcoI and XhoI, and ligated into the same sites of pET-6HGBP to make pET-6HGBP-ScFv.