Bunsei Kawakami

Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification

Magnetic particles are useful for simple and efficient nucleic acid extraction. To achieve fully automated nucleic acid extraction and purification using magnetic particles, a new method for operating magnetic particles, Magtration Technology, was developed. In this method, magnetic separation is performed in a specially designed disposable tip. This enables high recovery of magnetic particles with high reproducibility. The features of this technology are (i) a simple mechanism for process control and (ii) flexible software to enable adaptation to commercially available reagents. Automated instruments based on Magtration Technology were developed and used for nucleic acid extraction. Total DNA, total RNA and plasmids were purified by Magtration Technology at an efficiency comparable to that of manual methods.

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.

Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique

Specific interactions between transcription factors and cis‐acting DNA sequence motifs are primary events for the transcriptional regulation. Many regulatory elements appear to diverge from the most optimal recognition sequences. To evaluate affinities of a transcription factor to various suboptimal sequences, we have developed a new detection method based on the surface plasmon resonance (SPR) imaging technique. Transcription factor MafG and its recognition sequence MARE (Maf recognition elements) were adopted to evaluate the new method. We modified DNA immobilization procedure on to the gold chip, so that a double‐stranded DNA array was successfully fabricated. We further found that a hydrophilic flexible spacer composed of the poly (ethylene glycol) moiety between DNA and alkanethiol self‐assembled monolayers on the surface is effective for preventing nonspecific adsorption and facilitating specific binding of MafG. Multiple interaction profiles between MafG and six of MARE‐related sequences were observed by the SPR imaging technique. The kinetic values obtained by SPR imaging showed very good correlation with those obtained from electrophoretic gel mobility shift assays, although absolute values were deviated from each other. These results demonstrate that the double‐stranded DNA array fabricated with the modified multistep procedure can be applied for the comprehensive analysis of the transcription factor‐DNA interaction.

Construction and characterization of N-terminally truncated DNA polymerase from Thermus thermophilus

Abstract Various plasmids harboring the truncated DNA polymerase gene ( polA ) from Thermus thermophilus HB8 ( Tth polymerase) were constructed. The most thermostable Tth Δ NF 2 polymerase [the gene product of polA Δ NF 2, which lacked a 751-bp region (region flanked by initiation codon and Fsp I site in the polA gene)] was selected, and purified from the recombinant Escherichia coli . SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the Tth Δ NF 2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the Tth Δ NF 2 polymerase was 63% of that of the Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the Tth Δ NF 2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the Tth Δ NF 2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.

Nucleotide Sequence of the N-Acetylneuraminate Lyase Gene of Escherichia coli

Wehave established the sequence of a DNAfragment containing the Escherichia coli TV-acetylneuraminate lyase gene (nanA or npl). The coding sequence has an open reading frame of 297 amino acids. Amino acid compositions obtained by analysis of amino acids were mostly consistent with those calculated from the DNAsequence. The open reading frame contains a codonsequence of 15 amino acids deduced to be identical to the known Nterminal amino acid sequence of N-acetylneuraminate lyase. The first methionine was removedfrom the enzyme because the N-terminal amino acid was alanine.

Identification of regulatory motifs in the CHO genome for stable monoclonal antibody production.

Abstract Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.

Functional similarities of a thermostable protein-disulfide oxidoreductase identified in the archaeon Pyrococcus horikoshii to bacterial DsbA enzymes

We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH2-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH2-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.

The relaxation of specificity of BanI restriction endonuclease from Bacillus aneurinolyticus IAM 1077

Abstract The restriction endonuclease Ban I from Bacillus aneurinolyticus IAM 1077, which recognizes 5′-GGPyPuCC-3′ and cleaves between G and G within this sequence, has decreased substrate specificity at high nuclease concentrations. The relaxation of its specificity was enhanced during modified reactions: digestion of pBR322 DNA or lambda DNA in the presence of high glycerol and dimethyl-sulfoxide (DMSO) produced additional fragments in addition to the inherent fragments. Therefore, it is required to check the reaction conditions carefully for generation of inherent fragments.

CLONING AND EXPRESSION OF THE BAMHI RESTRICTION-MODIFICATION SYSTEM IN BACILLUS SUBTILIS

Abstract The genes of the GGATCC-specific Bam HI restriction-modification system of Bacillus amyloliquefaciens H have been cloned and expressed in Bacillus subtilis MT-2. B. subtilis MT-2 carrying the recombinant plasmid (pBamHIRM22) produced about 10-fold more Bam HI restriction endonuclease and Bam HI methylase than B. amyloliquefaciens H did. B. subtilis MT-2 (pBamHIRM22) restricted unmodified phage. Restriction and modification genes were stably maintained in B. subtilis MT-2 on one plasmid and the produced Bam HI endonuclease remained stable even in the stationary phase of the culture. Bam HI endonuclease from B. subtilis MT-2 (pBamHIRM22) had the same molecular weight and N-terminal amino acid sequence as that from B. amyloliquefaciens H.

Cloning and expression in Escherichia coli of the BanI and BanIII restriction-modification systems from Bacillus aneurinolyticus

Abstract The genes coding for the GGPyPuCC-specific ( Ban I) and ATCGAT-specific ( Ban III) restriction-modification systems of Bacillus aneurinolyticus IAM1077 were cloned and expressed in Escherichia coli using pBR322 as a vector. The plasmids carrying the Ban I and Ban III restriction-modification genes were designated pBanIRM8 and pBanIIIRM12, respectively. The restriction maps of these recombinant plasmids were constructed. These two plasmids were stably maintained in E. coli HB101. However, when E. coli JM109 was used as a host, pBanIIIRM12 was efficiently propagated but pBanIRM8 was not. The HB101 cells carrying only the restriction gene of Ban III were viable, but the Ban I restriction gene carrier could not form colonies on agar plates. The growth of bacteriophage λ was strongly restricted only in the F. coli HB101 cells harboring pBanIRM8. These facts indicate that the Ban I restriction enzyme is expressed and functions more efficiently than Ban I modification enzyme in E. coli .