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Y Kawarasaki

Publications and source records attributed to Y Kawarasaki.

7 recordsLinked to original sources
PubMed2000

Importance of disulfide bridge formation on folding of phospholipase D from Streptomyces antibioticus.

The effects of redox conditions on the folding of phospholipase D (PLD) of Streptomyces antibioticus were investigated. Although the enzyme was very stable even in the presence of 1.0 M guanidinehydrochloride (Gdn-HCl), the coexistence of dithiothreitol (DTT) and Gdn-HCl inactivated the enzyme completely. The inactivated enzyme recovered its activity by dialysis in which DTT was removed prior to Gdn-HCl, whereas its activity was not recovered when Gdn-HCl was removed prior to DTT. In vitro protein synthesis was used for further analyses of the folding process. Active PLD was synthesized in the absence of DTT. The activity increased as the protein synthesis proceeded. In contrast, inactive PLD was synthesized in the presence of DTT. The inactive PLD could not be effectively activated by simple removal of the reductant, while incubation with Gdn-HCl and subsequent removal of DTT followed by that of Gdn-HCl was a much more effective method for the synthesis of active enzymes. From these results, it is suggested that: (i) PLD contains disulfide bridge(s), which is (are) necessary for maintaining its tertiary structure, (ii) correct formation of the disulfide bridge(s) is a critical step in the early stage of the (re)folding process, and (iii) the disulfide bridge(s) further facilitate the folding process, resulting in the synthesis of the active enzymes with the correct structure.

Journal Article↗
PubMed1998

Phosphatase-immunodepleted cell-free protein synthesis system.

A cell-free protein synthesis system using wheat-germ extract was improved by a novel approach involving selective removal of endogenous phosphatase, which reduces both the duration and the rate of translation by hydrolyzing ATP and GTP, from the translational reaction. Immunodepletion of the phosphatases by the antibodies raised against the major one of the wheat-germ phosphatase isozymes removed 20-40% of ATP-hydrolysis activity from the wheat-germ extract, and thereby prolonged the reaction period of translation. Moreover, the condensation of the phosphate-immunodepleted extract by polyethylene glycol (PEG) precipitation and the addition of copper ions, which was known to inhibit phosphatase and nuclease activity, increased the protein synthesis more than two-fold compared with the reaction using control IgG-treated condensed extract.

Adenosine Triphosphate↗
PubMed1995

A long-lived batch reaction system of cell-free protein synthesis.

Several reaction conditions of cell-free protein synthesis such as temperatures, buffers, tRNAs, and creatine phosphate were intensively investigated and optimized to prolong protein synthesis and make it more efficiently in a batch system. As a result of these modifications, the protein synthesis reaction continued for 10 h so that about 30 micrograms of dihydrofolate reductase (DHFR) protein derived from Escherichia coli was synthesized in 1 ml of reaction mixture. In this improved system, translational reactions of other mRNAs such as rabbit beta-globin, Xenopus beta-globin, and tobacco mosaic virus RNA also continued for about 10 h. In addition, protein synthesis directed by uncapped dhfr mRNA containing a viral cap-independent translation initiation-mediating sequence continued for 10 h, resulting in the synthesis of 18 micrograms of DHFR protein per milliliter of reaction mixture.

Adenosine Triphosphate↗
PubMed1994

Prolonged cell-free protein synthesis in a batch system using wheat germ extract.

Reaction conditions of cell-free protein synthesis using wheat germ extract were examined to prolong the period of protein synthesis in a batch reaction. By optimizing conditions for ATP regeneration system involved in the cell-free system, protein synthesis continued about 4 hours, so that about 17 micrograms dihydrofolate reductase protein was obtained in 1 ml of a reaction mixture. It suggests that maintaining ATP concentration is the primary requirement for long-life cell-free protein synthesis.

Adenosine Triphosphate↗
PubMed1994

An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes.

Wheat-germ extract for cell-free protein synthesis was condensed with ultrafiltration membranes of which the molecular cut-off values were 10 kDa, 100 kDa, and 300 kDa. Reaction conditions of the cell-free system were optimized for the condensed extracts, which needed a higher concentration of creatine phosphate than the uncondensed one, probably due to the increased activity of degradation of ATP and GTP. By using the condensed extract and optimized reaction conditions, the rate of protein synthesis was increased 2- to 3-fold compared with using an uncondensed extract, and about 10-fold compared with conventional conditions. Condensation of the extract with the 300-kDa membrane showed the highest productivity, which was about 30 micrograms dihydrofolate reductase protein ml-1 h-1. The final amount of synthesized protein was one third of that of a continuous-flow cell-free (CFCF) system reported by Endo et al. [J. Biotechnol., 25, 221-230 (1992)] but the productivity was 5-fold higher than that obtained by the CFCF system.

Adenosine Triphosphate↗
PubMedDate not supplied

A trimmed viral cap-independent translation enhancing sequence for rapid in vitro gene expression.

We prepared a short (29 nucleotides) 5' UTR that enhanced cap-independent translation in a wheat germ translation system by trimming the tobacco etch virus 5' UTR. The trimmed sequence, designated as TE(37-65), was obtained from a conserved region among several potyviruses. The productivities of uncapped reporter mRNAs carrying the TE(37-65) sequence were comparable to those of capped counterparts, in that 5-20 microg of proteins were synthesized per 1 mL of translation reaction mixture. The ribosome that entered onto the TE(37-65) sequence precisely initiated polypeptide synthesis at the defined initiation codon, which ensures rapid and efficient protein truncation analyses. Moreover, the TE(37-65) sequence is short enough to be involved in a PCR primer, which allows a simple method for rapid gene expression, i.e., PCR amplification of a target gene and succeeding in vitro transcription and translation. As a demonstration, the rapid in vitro expression of rice cDNAs using the TE(37-65) sequence was also performed.

5' Untranslated Regions↗