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Kashiwa, Japan

Jackson K.,University of Florida | Kanamori T.,GeneFrontier Corporation | Ueda T.,Tokyo Medical University | Hugh Fan Z.,University of Florida
Integrative Biology (United Kingdom) | Year: 2014

Compared to cell-based protein expression, cell-free protein synthesis (CFPS) offers several advantages including a greater control over system additives. This control is further enhanced with a CFPS system called the Protein synthesis Using Recombinant Elements (PURE) system, which consists of 108 purified transcriptional and translational elements. With the PURE system, all elements are known, nuclease and protease activities are reduced, and the concentration of each element can be optimized for maximal protein expression. However, protein expression yield with this system is relatively low due to the consumption of nutrients and energy molecules as well as the accumulation of inhibitory byproducts in the batch format. To enhance protein expression with the PURE system, we developed a feeding solution that was optimized using a miniaturized fluid array device (μFAD) in a continuous-exchange cell-free (CECF) format. The device enabled (1) continuous supply of energy/nutrient molecules from the feeding solution to the reaction solution where protein synthesis occurred, and (2) simultaneous removal of inhibitory expression byproducts from the reaction solution to the feeding solution. Consequently, the synthesis yield of green fluorescent protein (GFP) increased 72.5-fold in comparison with the same reaction in the conventional batch format. © 2014 the Partner Organisations. Source


Kanamori T.,Tokyo Medical University | Kanamori T.,GeneFrontier Corporation | Fujino Y.,Tokyo Medical University | Fujino Y.,Mitsubishi Group | Ueda T.,Tokyo Medical University
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2014

Ribosome display utilizes formation of the mRNA-ribosome-polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA-ribosome-polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. © 2014 Elsevier B.V. Source


Trademark
GeneFrontier Corporation | Date: 2012-08-28

Proteins for scientific or medical research; Reagents for use in manufacture, separation, detection, measurement, identification or analysis of proteins for scientific or medical research; chemicals for scientific or medical research. Testing or research in the field of genetic or protein engineering; Testing or research on biotechnology and advice relating thereto; Testing, inspection or research of pharmaceuticals, cosmetics or foodstuff.


Patent
Genefrontier Corporation | Date: 2011-05-06

Provided is an antigen-binding protein prepared merely by a method of in vitro selection using the RNF8-FHA domain, which has no intramolecular disulfide bond and functions in cells as it is. One to four loops extending from the FHA domain are randomized, and a recognition site for a target molecule is artificially created on the FHA domain surface to construct an RNF8-FHA domain library. Using the library, an antigen-binding protein is efficiently selected in vitro.


Patent
Genefrontier Corporation and University of Tokyo | Date: 2011-06-23

The present invention provides a method of producing a maturated oligonucleotide library, including a step of obtaining a terminal-modified product of a maturation target oligonucleotide library, including adding a tag sequence to the 5 terminus of the maturation target oligonucleotide library and an arrest sequence, which stalls translation elongation on a ribosome, to the 3 terminus of the maturation target oligonucleotide library, a step of transcribing the terminal-modified sequence product to give a transcript, and a step of in vitro translation for translating the transcript in vitro, wherein the maturation target oligonucleotide library is a random oligonucleotide library.

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