June-Hyung Kim

A novel sensor platform based on aptamer-conjugated polypyrrole nanotubes for label-free electrochemical protein detection.

We first present a simple yet versatile strategy for the functionalization of polymer nanotubes in a controlled fashion. Carboxylic‐acid‐functionalized polypyrrole (CPPy) nanotubes were fabricated by using cylindrical micelle templates in a water‐in‐oil emulsion system, and the functional carboxyl groups were effectively incorporated into the polymer backbone during the polymerization by using pyrrole‐3‐carboxylic acid (P3CA) as a co‐monomer without a sophisticated functionalization process. It was noteworthy that the chemical functionality of CPPy nanotubes was readily controlled in both qualitative and quantitative aspects. On the basis of the controlled functionality of CPPy nanotubes, a field‐effect transistor (FET) sensor platform was constructed to detect specific biological entities by using a buffer solution as a liquid‐ion gate. The CPPy nanotubes were covalently immobilized onto the microelectrode substrate to make a good electrical contact with the metal electrodes, and thrombin aptamers were bonded to the nanotube surface via covalent linkages as the molecular recognition element. The selective recognition ability of thrombin aptamers combined with the charge transport property of CPPy nanotubes enabled the direct and label‐free electrical detection of thrombin proteins. Upon exposure to thrombin, the CPPy nanotube FET sensors showed a decrease in current flow, which was probably attributed to the dipole–dipole or dipole–charge interaction between thrombin proteins and the aptamer‐conjugated polymer chains. Importantly, the sensor response was tuned by adjusting the chemical functionality of CPPy nanotubes. The efficacy of CPPy nanotube FET sensors was also demonstrated in human blood serum; this suggests that they may be used for practical diagnosis applications after further optimization.

High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis

Staphylokinase is a promising blood-clot dissolving agent for the treatment of patients suffering from a heart attack. It would be desirable to produce this protein in large quantities for biochemical characterization and clinical trials. Production of intact, biologically active staphylokinase from bacterial expression systems has been a challenge because of N-terminal microheterogeneity, plasmid instability, or low-production yield. By using a seven-extracellular-protease deficient Bacillus subtilis strain, WB700, intact staphylokinase can be produced via secretion. However, native staphylokinase gene (sak) in a high-copy number plasmid was found to be unstable in B. subtilis. To optimize the production and the stability of the expression vectors, both the promoter and the signal sequence of sak were replaced by B. subtilis promoters (P43, a constitutively expressed promoter; Pamy, a stationary-phase promoter; and PsacB, a sucrose-inducible promoter) and the levansucrase-signal sequence, respectively. This overcame the plasmid instability problem. To enhance transcription from the sacB promoter, degQ encoding a transcriptional activator for sacB and other protease genes was also installed in the expression vector. The use of WB700 as the expression host allowed enhanced production of staphylokinase from the sucrose-inducible plasmid without causing any obvious degradation of staphylokinase. Both the P43 and PsacB (with DegQ) promoters worked well. Over 90% of staphylokinase synthesized can be secreted effectively. With the optimization of both the culture media and the fermentation conditions, production of staphylokinase reached a level of 337 mg/L, and staphylokinase could be purified to homogeneity by a simple three-step purification scheme. Secreted staphylokinase did not show any N-terminal heterogeneity. This presents an attractive system for the production of staphylokinase in both high quality and quantity.

Biomolecular detection with a thin membrane transducer

We present a thin membrane transducer (TMT) that can detect nucleic acid based biomolecular reactions including DNA hybridization and protein recognition by aptamers. Specific molecular interactions on an extremely thin and flexible membrane surface cause the deflection of the membrane due to surface stress change which can be measured by a compact capacitive circuit. A gold-coated thin PDMS membrane assembled with metal patterned glass substrate is used to realize the capacitive detection. It is demonstrated that perfect match and mismatch hybridizations can be sharply discriminated with a 16-mer DNA oligonucleotide immobilized on the gold-coated surface. While the mismatched sample caused little capacitance change, the perfectly matched sample caused a well-defined capacitance decrease vs. time due to an upward deformation of the membrane by a compressive surface stress. Additionally, the TMT demonstrated the single nucleotide polymorphism (SNP) capabilities which enabled a detection of mismatching base pairs in the middle of the sequence. It is intriguing that the increase of capacitance, therefore a downward deflection due to tensile stress, was observed with the internal double mismatch hybridization. We further present the detection of thrombin protein through ligand-receptor type recognition with 15-mer thrombin aptamer as a receptor. Key aspects of this detection such as the effect of concentration variation are investigated. This capacitive thin membrane transducer presents a completely new approach for detecting biomolecular reactions with high sensitivity and specificity without molecular labelling and optical measurement.

Rational design of modular allosteric aptamer sensor for label-free protein detection.

An aptamer can be redesigned to new functional molecules by conjugating with other oligonucleotides. However, it requires experimental trials to optimize the conjugating module with the sensitivity and selectivity toward a target. To reduce these efforts, we report rationally-designed modular allosteric aptamer sensor (MAAS), which is composed of coupled two aptamers and the regulator. For label-free protein detection, the protein-aptamer was conjugated with the malachite green (MG) aptamer for signaling. The MAAS additionally has the regulator domain which is designed to hybridize to a protein binding domain. The regulator makes MAAS to be inactive by destructing the original structure of the two aptamers. However, its conformation becomes active by dissociating the hybridization from the protein recognition signal, thereby inducing the binding of MG emitting the enhanced fluorescence. The design of regulator is based on the thermodynamic energy difference by the RNA conformational change and protein-aptamer affinity. Here we first demonstrated the MAAS for hepatitis C helicase and replicase. The target proteins were detected up to 250nM with minimized blank signals and displayed high specificities 10-fold greater than in non-specific proteins. The MAAS provides valuable tools that can be adapted to a wide range of configurations in bioanalytical applications.

Selection of peptides for lipopolysaccharide binding on to epoxy beads and selective detection of Gram-negative bacteria

Lipopolysaccharide (LPS)-binding peptides were enriched by using epoxy beads as a novel support to immobilize LPS for a phage displayed peptide library screening. The sequence of Phe-Ala-Pro-Trp (FAPW) was the most significant consensus motif of 10 selected clones, and Pro-Phe (PF) was the key dipeptide for binding at the apex of the loop to form a characteristic structure of CXXPFXXXC. Moreover, AWLPWAK, one of the highly conserved heptamer peptides, could detect specifically Gram-negative bacteria via a whole cell binding test at 106 cells ml−1.

Optimization of staphylokinase production inBacillus subtilis using inducible and constitutive promoters

Staphylokinase (SAK) was produced inB. subtillis using two different promoter systems,i.e. the P43 andsacB promoters. To maximize SAK expression inB. subtilis, fermentation control strategies for each promoter were examined. SAK, under P43, a vegetative promoter transcribed mainly by σB containing RNA polymerase, was overexpressed at low dissolved oxygen (D.O.) levels, suggesting that thesigB operon is somewhat affected by the energy charge of the cells. The expression of SAK at the 10% D.O. level was three times higher than that at the 50% D.O. level. In the case ofsacB, a sucrose-inducible promoter, sucrose feeding was used to control the induction period and induction strength. Since sucrose is hydrolyzed by two sucrose hydrolyzing enzymes in the cell and culture broth, the control strategy was based on replenishing the loss of sucrose in the culture. With continuous feeding of sucrose, WB700 (pSAKBQ), which contains the SAK gene undersacB promoter, yieldedca. 35% more SAK than the batch culture. These results present efficient promoter-dependent control strategies inB. subtilis host system for foreign protein expression.

Bacterial Surface Display of a Co-Factor Containing Enzyme, ω-Transaminase from Vibrio fluvialis Using the Bacillus subtilis Spore Display System

To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.

In vitro selection of sialic acid specific RNA aptamer and its application to the rapid sensing of sialic acid modified sugars

Sialic acids (SAs) are located on the terminal positions of glycan on a cell surface, which play important role in the spread and metastasis of cancer cells and infection of pathogen. For their detection and diagnosis, the finding of SA specific ligand is an essential prerequisite. Here, RNA aptamer for N‐acetylneuraminic acid (Neu5Ac), a representative of SAs, with the high affinity of 1.35 nM and the selectivity was screened by in vitro selection method. The strong binding of the screened aptamer was enough to protect the hydrolysis of Neu5Ac by neuraminidase with the stoichiometry of 1:1 molar ratio. For the rapid detection of SAs, the RNA aptamer was further engineered to the aptazyme sensor by conjugating with a ribozyme following the characterization of selected aptamer by RNase footprinting assay. Without additional desialylation, modification, or/and purification processes, the aptazyme indicated high catalytic activities in the presence of Neu5Ac over 20 µM in several minutes. Also, we observed that the aptazyme sensor shows high sensitivities to Neu5Ac‐conjugated sugars as well as Neu5Ac monomer, but not in non‐Neu5Ac modified sugars. The aptamer for Neu5Ac can support valuable tools in a wide range of bioanalytical applications as well as biosensors. Biotechnol. Bioeng. 2013; 110: 905–913.

Construction of spore mutants of Bacillus subtilis for the development as a host for foreign protein production

For the development of Bacillus subtilis as a host for foreign protein synthesis, three types of sigma factor deleted mutants (spoIIAC, spoIIIG and spoIIIC) were constructed by antibiotic marker insertion using plasmid vector-mediated method or LFH (Long Flanking Homology)-PCR. Mother cell specific sigma factor mutants of B. subtilis (σK), B. subtilis DB104 ΔspoIIIC (kmr)::pMK101, had two to three times higher subtilisin activity than the wild type DB104::pMK101. Subtilisin expression by the other two mutants, B. subtilis DB104 ΔspoIIAC (kmr)::pMK101 and DB104 ΔspoIIIG (kmr)::pMK101, which are pre-spore specific sigma factor (σF and σG) deleted strains, was similar to, or less than that of the wild type.

Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry : A tool for functional genomics

For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lip A and lip B from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.