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Engineered organisms with an expanded genetic code have attracted much attention in chemical and synthetic biology research. In this work, engineered bacterial organisms with enhanced unnatural amino acid (UAA) uptake abilities were developed by screening periplasmic binding protein (PBP) mutants for recognition of UAAs. A FRET-based assay was used to identify a mutant PBP (LBP-AEL) with excellent binding affinity ( Kd ≈ 500 nM) to multiple UAAs from 37 mutants. Bacterial cells expressing LBP-AEL showed up to 5-fold enhanced uptake of UAAs, which was determined by genetic incorporation of UAAs into a green fluorescent protein and measuring UAA concentration in cell lysates. To the best of our knowledge, this work is the first report of engineering cellular uptake of UAAs and could provide an impetus for designing advanced unnatural organisms with an expanded genetic code, which function with the efficiency comparable to that of natural organisms. The system would be useful to increase mutant protein yield from lower concentrations of UAAs for industrial and large-scale applications. In addition, the techniques used in this report such as the sensor design and the measurement of UAA concentration in cell lysates could be useful for other biochemical applications.

Hsp70 is known to directly bind to Bax for suppression of apoptosis. However, mechanisms on how Bax is dissociated from its complex with Hsp70 during apoptosis remain largely unknown. In the current study, we developed the efficient fluorescence resonance energy transfer (FRET) system which consisted of Hsp70-YFP and fluorescent amino acid (ANAP)-incorporated Bax, which was generated by using genetic code expansion technology, and applied the FRET system to elucidate mechanisms on how apoptosis-inducing substances dissociate Bax from Hsp70. Time-dependent analysis of single live cell images showed that Bax activators binding to Bax trigger sites inhibited the Bax-Hsp70 interaction but a Bax activator, which blocks phosphorylation of S184 via binding to the C-terminal S184 site, did not affect this interaction. Additionally, an inhibitor for Hsp70-Hsp40 interaction blocked the Bax-Hsp70 interaction. Furthermore, p53 activators promoted the dissociation of Bax from Hsp70 by reactivating p53 which disrupted the Bax-Hsp70 interaction. We also found that death ligands and a Bcl-2 inhibitor enhanced the dissociation of Bax from Hsp70 by activating activator BH3-only proteins. Results from this effort suggest that FRET systems consisting of the ANAP-incorporated protein and the YFP fusion protein will be valuable tools to gain an understanding of other types of protein-protein interactions.

Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators.

To gain insight into factors that lead to dissociation of Bax from a complex with Hsp70 during apoptosis, we recently constructed a fluorescence resonance energy transfer (FRET) system composed of the Hsp70-YFP (YFP=yellow fluorescent protein) fusion protein and fluorescent amino acid (ANAP=6-acetyl(naphthalen-2-ylamino)-2-aminopropanoic acid)-containing Bax (Bax-ANAP), which was produced by using the genetic code expansion technique. In the current study, the FRET system was employed to elucidate how brefeldin A (an endoplasmic reticulum stress inducer), chlorpromazine and apoptozole (lysosomal membrane destabilizers), bafilomycin A1 (an inhibitor of lysosomal acidification) as well as raptinal and Az-TPP-O3 (mitochondria-targeted apoptosis inducers) affect the interaction between Bax and Hsp70. Analyses of single live cell images together with results of co-immunoprecipitation assays reveal that brefeldin A, chlorpromazine, and apoptozole promote dissociation of the Bax/Hsp70 complex through activation of the activator BH3-only protein. However, the results show that bafilomycin A1, raptinal, and Az-TPP-O3 have no influence on the interaction of Bax with Hsp70. The combined observations made in the current and previous studies demonstrate that the FRET system consisting of Bax-ANAP and Hsp70-YFP is highly useful to understand apoptotic processes associated with the Bax-Hsp70 interaction.

Amino acids are essential nutrients that are not only used as protein building blocks but are also involved in various biochemical processes and in the development of human diseases. Quantitative analysis of amino acids in complex biological samples is an important analytical process used for understanding amino acid biochemistry and diagnosis of human diseases. In this study, a protein sensor based on fluorescence resonance energy transfer (FRET) was designed for the quantitative analysis of L-Met, in which a fluorescent unnatural amino acid (CouA) and YFP were used as a FRET pair. A natural Met-binding protein (MetQ) was chosen as a sensor protein, and CouA and YFP were incorporated into the protein by genetic code expansion technology and genetic fusion. Among the four sites screened for CouA incorporation into MetQ, R189 was selected as the best site for L-Met sensing. The sensor protein (YFP-MetQ-R189CouA) showed a large FRET signal change (2.7-fold increase) upon L-Met binding. To improve amino acid specificity of the sensor protein, the ligand-binding site was engineered, and the mutant sensor (YFP-MetQ-R189CouA-H88F) with the H88F mutation was identified, which showed no FRET signal change with D-Met and L-Gln at 50 μM concentration and retained the maximum FRET signal change with L-Met. The optimized sensor protein was evaluated for biochemical applications. L-Met concentration in FBS and optical purity in a mixture of D- and L-Met were successfully determined. Because L-Met is biochemically important owing to its involvement in cancer cell growth and autophagy, the sensor protein would be useful for quantitative analysis of L-Met in a complex biological sample. In addition, the design strategy used in this study can be applied to other small molecule-binding proteins for the development of protein sensors for important biomolecules.

Large DNA molecules were visualized with novel fluorescent protein–DNA binding peptides (FP–DBPs). We constructed FP–DBPs by linking fluorescent protein to the N- or C-terminal of one or two peptides. We designed these DNA binding peptides from various DNA binding motifs such as oligo-lysine (K) and Lys-Trp (KW) repeats, TPKRPRGRPKK from high mobility group (HMG) chromosomal protein, and Ser-Pro-Arg-Lys (SPRK) from histone protein. We demonstrated the use of FP–DBP to stain large DNA molecules, and then analysed the fluorescence brightness and their binding affinity to double stranded DNA. This investigation provided HMG-tagged FP–DBP as the best DNA staining reagent in terms of fluorescence intensity, signal-to-noise ratio, and DNA binding affinity (Kd = 586 nM). Furthermore, we measured elongation of FP–DBP-stained DNA molecules tethered on the surface in order to evaluate FP–DBP-induced structural deformation.

FRET sensors based on fluorescent proteins have been powerful tools for probing protein–protein interactions and structural changes within proteins. However, they are intrinsically limited by their large size and the requirement for N- or C-terminal fusions. In this report, a FRET-based sensor was developed by incorporating a fluorescent unnatural amino acid into glutamine-binding protein (GlnBP), which formed a FRET pair with green fluorescent protein (GFP). GFP was fused to the N-terminus of GlnBP, and the fluorescent unnatural amino acid was incorporated into the site for N138 in GlnBP. FRET sensors were tested that contained linkers of different length between GFP and GlnBP, and assay conditions were optimized by changing the pH and salt concentration of the assay buffer. Under optimal conditions, the best sensor protein produced a 1.9-fold increase in the FRET ratio upon L-Gln binding, whereas either no change or minimal change was seen using other amino acids, including the other 19 natural amino acids and D-Gln. This novel design strategy for FRET sensors overcomes the limitations of current FRET sensors, which require the use of two fluorescent proteins. Consequently, our strategy may prove useful for investigating protein–protein interactions and for probing changes in protein conformation.

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance.

The genetic incorporation of unnatural amino acids (UAAs) into proteins has been a useful tool for protein engineering. However, most UAAs are expensive, and the method requires a high concentration of UAAs, which has been a drawback of the technology, especially for large-scale applications. To address this problem, a method to recycle cultured UAAs was developed. The method is based on recycling a culture medium containing the UAA, in which some of essential nutrients were resupplemented after each culture cycle, and induction of protein expression was controlled with glucose. Under optimal conditions, five UAAs were recycled for up to seven rounds of expression without a decrease in expression level, cell density, or incorporation fidelity. This method can generally be applied to other UAAs; therefore, it is useful for reducing the cost of UAAs for genetic incorporation and helpful for expanding the use of the technology to industrial applications.

Protein–nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein–DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein–DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.

Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.

This paper proposes a new measurement system for measuring the refractive power of spherical and sphero-cylindrical lenses with a six-point light source, which is composed of a light emitting diode and a six-hole pattern aperture, and magnification ellipse fitting method. The position of the six light sources is changed into a circular or elliptical form subjected to the lens refractive power and meridian rotation angle. The magnification ellipse fitting method calculates the lens refractive power based on the ellipse equation with magnifications that are the ratios between initial diagonal lengths and measured diagonal lengths of the conjugated light sources changed by the target lens. The refractive powers of the spherical and sphero-cylindrical lenses certified in the Korea Research Institute of Standard and Science were measured to verify the measurement performance. The proposed method is estimated to have a repeatability of +/-0.01 D and an error value below 1%.

This research proposes a new non-invasive and non-contact diffuse optical tomography (DOT) system for imaging of blood vessel with CW near-infrared (NIR) light. A DOT system can measure the hemoglobin distribution inside tissue by detecting the NIR light emitting from the tissue. The proposed non-contact DOT system is based on the scanning system which offers the advantage of performing non-invasive, real time, and cost-effective measurement. The non-contact detection system is constructed with dual NIR CCD cameras and multiple lens system. This non-contact DOT system detects the light emitting from slab geometry. A Rytov method is used for reconstructing images from intensity information of CCD. The non-contact DOT system is experimentally tested with phantom consisting of paraffin wax and Intralipid. To improve the performance of the non-contact DOT system, modifications of inverse algorithm and scanning method are required.

Four unnatural amino acids (UAAs) were synthesized from their corresponding α-keto acids by using glutamine:phenylpyruvate aminotransferase (GlnAT) from Thermus thermophilus HB8. The enzyme efficiently catalyzed the conversion of the unnatural substrates, with little decrease in its activity compared to that shown toward its natural substrates. The α-keto acids were converted into the corresponding amino acids in cells and then directly incorporated into a protein with high efficiency and fidelity. Because α-keto acids have no chiral center and are synthetically readily accessible, this method will further enhance the genetic incorporation technology and may present a new approach to the preparation of optically pure UAAs. Considering the substrate promiscuity of the GlnAT observed in the study, this method can be applied to the genetic incorporation of other UAAs derived from Tyr or Phe, which constitute approximately 50% of the successfully incorporated UAAs. In addition, this study provides an impetus for the development of an autonomous unnatural organism that can synthesize and incorporate UAAs.