Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Complementary Cell-Free Translational Assay for Quantification of Amino Acids.

In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signal-generating protein. This method harnesses amino acid-deficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable biofluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.

Cell-free synthesis of functional phospholipase A1 from Serratia sp.

BACKGROUND: Phospholipase A1 is an enzyme that hydrolyzes phospholipids at the sn-1 position. It has potential applications across diverse fields including food, pharmaceutical, and biofuel industries. Although there has been increasing interest in the use of phospholipase A1 for degumming of plant oils during biodiesel production, production of recombinant phospholipase A1 has been hampered by low efficiency of gene expression and its toxicity to the host cell. RESULTS: While expression of phospholipase A1 in Escherichia coli resulted in extremely low productivity associated with inhibition of transformed cell growth, drastically higher production of functional phospholipase A1 was achieved in a cell-free protein synthesis system where enzyme expression is decoupled from cell physiology. Compared with expression in E. coli, cell-free synthesis resulted in an over 1000-fold higher titer of functional phospholipase A1. Cell-free produced phospholipase A1 was also used for successfully degumming crude plant oil. CONCLUSIONS: We demonstrate successful production of Serratia sp. phospholipase A1 in a cell-free protein synthesis system. Including the phospholipase A1 investigated in this study, many industrial enzymes can interfere with the regular physiology of cells, making cellular production of them problematic. With the experimental results presented herewith, we believe that cell-free protein synthesis will provide a viable option for rapid production of important industrial biocatalysts.

Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons.

The genetic code in most organisms codes for 20 proteinogenic amino acids or translation stop. In order to encode more than 20 amino acids in the coding system, one of stop codons is usually reprogrammed to encode a non-proteinogenic amino acid. Although this approach works, usually only one amino acid is added to the amino acid repertoire. In this study, we incorporated non-proteinogenic amino acids into a protein by using a sense codon. As all the codons are allocated in the universal genetic code, we destroyed all the tRNA(Arg) in a cell-free protein synthesis system by using a tRNA(Arg) -specific tRNase, colicin D. Then by supplementing the system with tRNACCU , the translation system was partially restored. Through this creative destruction, reprogrammable codons were successfully created in the system to encode modified lysines along with the 20 proteinogenic amino acids.

Synthesis and Evaluation of Neuroprotective Selenoflavanones.

The physicochemical properties and antioxidant activity of a molecule could be improved by the substitution of an oxygen atom in a molecule with selenium. We synthesized selenoflavanones and flavanones to evaluate their neuroprotective effects. The selenoflavanones showed improved physicochemical properties, suggestive of the ability to pass through the blood-brain barrier (BBB). They showed in vitro antioxidant effects against hydrogen peroxide, and did not result in severe cytotoxicity. Moreover, infarction volumes in a transient ischemia mouse model were significantly reduced by the selenoflavanone treatments.

Patterns of subsequent progression of localized retinal nerve fiber layer defects on red-free fundus photographs in normal-tension glaucoma.

PURPOSE: To investigate patterns of subsequent progression of localized retinal nerve fiber layer (RNFL) defects and to quantify the extent of progression in normal-tension glaucoma (NTG) patients. METHODS: Thirty-three eyes of 33 consecutive NTG patients who had shown continuous progression of localized RNFL defect on serial red-free fundus photographs were selected for the study. Patterns of subsequent progression of localized RNFL defects were categorized, and extents of progression were quantified. Serial evaluations of disc stereophotographs and visual fields were also performed to detect progression. RESULTS: The most common pattern was continuous widening of the defect towards the macula (n = 11, 33.3%) followed by sharpening of the defect border after widening of the defect towards the macula (n = 5, 15.2%), continuous widening of the defect away from the macula (n = 2, 6.1%), and deepening of the defect after appearance of a new defect (n = 2, 6.1%). Four eyes (12.1%) simultaneously showed two patterns of subsequent progression. In 13 eyes that showed continuous widening of the defect, subsequent angular widening towards the macula and away from the macula were 9.2 ± 6.0° (range, 1.1° to 24.4°; n = 11) and 5.2 ± 4.9° (range, 0.3° to 11.3°; n = 2), respectively. Thirty-two eyes showed no progression of optic disc cupping. Out of the 21 eyes in which Humphrey central 30-2 threshold visual field tests were performed after progression of RNFL defects, 15 eyes showed no deterioration in the visual field. CONCLUSIONS: There were nine patterns of subsequent progression of localized RNFL defects. Among them, continuous RNFL loss proceeding temporally was the most common one. Initial progression of the defect proceeded temporally, especially in the defect located at the inferior fundus, might be at a risk of further RNFL loss temporally.

The effects of supplementing specific amino acids on the expression of elastin-like polypeptides (ELPs).

Elastin-like polypeptides (ELPs) made from the repeating pentapeptides (Val-Pro-Gly-Xaa-Gly) are protein based biopolymers that contain useful properties, including the ability to self-assemble, biocompatibility, and stimuli sensitivity. However, due to the repeated consumption of specific amino acids, long ELPs generally have low expression yields in in vitro and in vivo systems. This is because of the lack of specific amino acids during the translation process. In this study, ELP fusion proteins of various lengths were prepared by recursive directional ligation (RDL) and expressed in a cell-free protein synthesis system. By measuring TCA-precipitated radioactivity with a liquid scintillation counter, their expression profiles were investigated. The expression levels of an ELP fusion protein were improved by almost 2-fold by adding specific amino acids. Additionally, we determined that the amount of increase in expression levels depends on the length of the ELPs. This study suggests a useful strategy to improve the yield of longer repetitive polypeptides such as ELPs or silk-like polypeptides (SLPs).

Thermal injury induces heat shock protein in the optic nerve head in vivo.

PURPOSE: To investigate the induction of heat shock protein (Hsp)70 in the optic nerve head by localized laser application in transpupillary thermotherapy (TTT). METHODS: TTT was performed on the right eye of Norwegian brown rats with an 810-nm diode laser installed on a slit lamp biomicroscope. The laser was aimed at the center of the optic nerve head with a 50-microm spot size. Various exposures (range, 60-200 mW) were used with an exposure duration of 60 seconds, and the various exposure durations (range, 1-5 minutes) were used with a power of 100 mW. Twenty hours after laser irradiation, immunohistochemical staining and Western blot analyses were performed. For morphologic analysis of the optic nerve head, confocal scanning laser ophthalmoscopy and scanning electron microscopy were performed. RESULTS: In the control eyes, Hsp70 was detected minimally in the optic nerve tissues by immunohistochemistry. After TTT, Hsp70 in the optic nerve tissue was induced more than in the control eyes. By Western blot, Hsp70 expression was found to increase progressively after TTT as the power was increased, but it also decreased slightly at powers >140 mW. The optimal setting of TTT without tissue damage was determined to be 100 mW for 60 seconds. CONCLUSIONS: Transpupillary laser irradiation of the optic nerve head induces Hsp70 expression. This result can be applied to the neuroprotective experiments in glaucoma by enhancement of a natural cytoprotective stress response.

Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system.

We developed a cell-free protein synthesis system that produces more than 1mg/ml of recombinant proteins in two hours. A basal system that supports the stable maintenance of ATP and amino acids was constructed by using high concentrations of CP (100 mM) and amino acids (3 mM). Approximately 0.6 mg/ml of protein was produced during the batch incubation of the basal system. We found that the accumulation of inorganic phosphate reduces the concentration of free magnesium ions and that there exists a critical concentration of magnesium at which the protein synthesis is halted. Based on this finding, we attempted to extend the duration of the protein synthesis by keeping the magnesium concentration sufficiently high throughout the reaction period. The protein synthesis reaction continued for at least 2 h when the reaction was repeatedly supplemented with magnesium, and approximately 1.2 mg/ml of active CAT or GFP was produced. The simple, fast, and highly productive cell-free protein synthesis system described herein should offer a versatile platform for the preparation of protein molecules in various post-genomic efforts.

Neuroprotective effect of memantine in a rabbit model of optic nerve ischemia.

The purpose of this study was to evaluate the neuroprotective effect of memantine, a N-methyl-D-aspartate antagonist, in an experimental optic nerve ischemia. Endothelin-1 (ET-1) in a dosage of 0.1 microg/day was delivered to the perineural region of the anterior optic nerve by osmotically driven minipumps for 8 weeks in 10 rabbits. In 5 rabbits, 1 mg/kg memantine was administered concurrently by intramuscular injection once a daily. Morphologic optic nerve head changes were monitored with a confocal scanning laser ophthalmoscope. Multivariate statistical analysis showed a significant change in topometric parameters (cup area, cup depth and rim volume), indicating an increase in optic nerve head cupping and a decrease of neural rim volume in the ET-1 administered eyes (P < 0.0001). In rabbits where memantine was given concurrently with ET-1, no significant change in topometric parameters was observed after ET-1 administration (P = 0.78). The current results suggest that memantine has a neuroprotective effect in optic nerve ischemia. Memantine may potentially be useful in the management of various ischemic disorders of the optic nerve, including glaucoma.