Preparation of a plasmid encoding GBP-ProG fusion protein

We created a vector capable of producing the fusion protein, including gold binding polypeptide (GBP) and Protein G (ProG).The GBP specifically bound against the structure of goldsurface was genetically fused to the N-terminus of protein G, and asix-histidine (6His) tag was fused to the N-terminus of the GBP foreasy purification of the fusion proteins. The DNA fragments encoding GBP-ProGalso include anderson promotor(J23100), ribosome binding site(B0034), and double terminators (B0010, B0012). Whole amplifying procedure of the constructwascarried outfromIntegrated DNA Technologies (IDT).

We conducted A-tailing process at the end of our gene fragment for TA cloning. A-tailing is one of enzymatic methodsto add a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA. Tailing is a general procedure to prepare a T-vector or to A-tail a PCR product amplified with high-fidelity polymerase for use in TA cloning.

The A-tailedgene fragment was ligated with the TA vector at room temperature for 2 hours.Then, the vector was delivered toE. coli DH5-alpha competent cell. The bacteria were grown in LB plate supplemented with X-gal for selectionbacterial cells containing recombinant DNA.

Table 1. Reaction conditions for restriction enzymes and ligation

We picked 4 white colonies from the platesthe cells were grown in LB broth with 0.1mM of ampicillin at 37°C and 220 rpm. The expanded vectors wereisolated through DNA mini prep kit. The TA-GBP-ProG vector wasincubatedwith XbaI and SpeI at 37°C for 2 hours. The DNA fragment parts separated from the plasmid backbone were purified using gel electrophoresis and ligated with pSB1C3 vector.

Figure 1. Gel electrophoresis of XbaI and SpeI-digested plasmid from pSB1C3 and TA-GBP-ProG

The ten microliter of the ligation sample was transformed to 100μl of E. coli DH5-alpha. Then, theovernight culture wasperform in LB plate including chloramphenicol. We picked 10 colonies from the plate and an additional overnight culture were achieved at 37°C and 220 rpm.The amplified plasmid was purified through DNA mini prep kit. We checked appropriate insertion of the construct in the vector by gel electrophoresis after reaction with XbaI and SpeI to verify the exact band scale. Figure 2 showed that lane1, 4, 5, 6 and 7 is proper orientation producing of pSB1C3-GBP-ProG.

Figure 2. Gel electrophoresis of XbaI and SpeI-digested plasmid from bacteria transformed with the ligation mixture to generate pSB1C3-GBP-ProG

Plasmid sequence was confirmed by BIOFACT™ sequencing analysis service. We have confirmed that wholesequence of the engineered parts were correct. Primer sequence was as follows.

VF2(Forward): 5’-TGCCACCTGACGTCTAAGAA-3’

VR(Reverse): 5’-ATTACCGCCTTTGAGTGAGC-3’

Figure 3. Sequencing results from BIOFACT™ sequencing analysis service

Amplification and purification of GBP-ProG fusion Protein

After IPTG-induced expression, the entire culture was centrifuged and discarded the upper layer. The remaining cell pellet wassuspended in a resin binding bufferand the cells werelysed at 4 ℃ by sonication protocol. The lysed solution was centrifuged at 4 ℃ for 30 minutes at 13,000 rpm to obtain the supernatant which contains whole proteins from the bacterial cells. With the TALON metal affinity resin, the purified proteins were confirmed by SDS-PAGE, and then quantified using the Bradford protein assay to dilute to 1mg/ml with a phosphate buffered solution (PBS).

Figure 4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of amplified recombinant GBP-ProG and total proteins. M, molecular weight marker; T,total protein; B,aliquot of resin binding residue; W1,aliquot of 1st washing residue; W2,aliquot of 2nd washing residue;W3,aliquot of 3rd washing residue;E,purified GBP-ProG

Fabrication of interdigitated array gold electrode

We preparedan interdigitated array gold electrode to develop a novel electrochemical immunosensor for monitoring heart failure patients. Interdigitated array(IDA) chip with three-electrode system of reference, counter,and working electrode, fabricated by standard photolithographyand lift-off process. Chrome (50 Åm thick) and gold (100 Åm thick) weresequentially deposited on a cyclic olefin copolymer (COC)wafer by sputtering. Thephotoresist (PR) wasspin-coated on the COC wafer at 4000 rpm, then baking at 90 °C for90 s. PR exposed to UV light(2.0 s, 16 mW/cm2) via a photomask after, and then developed. Gold and chrome was removed by etchant solution. The PR wasremoved by washing with acetone.

Preparation of electrochemical immunochip

The electrochemical immunochip was fabricated by assembling the IDA array gold electrode with the bare COC wafer that inlet/outlet holes (1 mm in diameter) were punched for interconnection of a channel. To stick two parts together, we used a double-sided tape (thickness: 50 μm) wounded at direction of the channel, followed by generation of 50 μm gap between the upper and lower wafer.

Electrochemical immunoassay

After fabricating the immunochip, cyclic voltammograms were measured with the injection of PBS as a background signal. Then, the GBP-ProG, BSA, anti-NTproBNP, BSA, NTproBNP, AP-antiNTproBNP and PAPP were sequentially injected with PBS washing between each step. For electrochemical detection of PAP, cyclic voltammograms were obtained by the oxidation peak current proportionally to its concentration during electrooxidation into PQI that can be easily reduced.

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