[Show abstract][Hide abstract]ABSTRACT: Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.
[Show abstract][Hide abstract]ABSTRACT: 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 NH(2)-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 NH(2)-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.
[Show abstract][Hide abstract]ABSTRACT: Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called the polyhedron in which progeny virions are occluded. The virion structural protein, VP3, is implicated in the occlusion of viral particles into polyhedra. In this study, we determined the amino acid sequence of VP3 required for occlusion of viral particles into polyhedra and proposed that this sequence could be used as an immobilization signal to direct the stable incorporation of foreign proteins into polyhedra. A large-scale survey revealed that the immobilization signal could, in fact, direct the incorporation of a variety of human proteins into polyhedra. Immune reactivity and protein-protein interactions were detected on the surface of polyhedra containing immobilized foreign proteins, and these particles were shown to be highly stabilized against dehydration. We showed that these particles could be arrayed onto a glass slide by standard spotting and laser manipulation methods. Thus, this approach is well suited for protein expression, purification, and the development of protein microarrays.
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Feb 2005 · Nucleic Acids Research
[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.
[Show abstract][Hide abstract]ABSTRACT: 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.
No preview · Article · Feb 2001 · Journal of Bioscience and Bioengineering
[Show abstract][Hide abstract]ABSTRACT: The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.
Full-text · Article · Dec 1997 · Applied and Environmental Microbiology
[Show abstract][Hide abstract]ABSTRACT: Serratia liquefaciens was screened as a host strain for effective gene expression and easy purification of the target protein. A model gene, N-acetylneuraminate lyase gene (nanA), fused with the promoter region of Escherichia coli lac operon successfully overproduced the protein independently from the inducer. Since S. liquefaciens grew at lower temperature than E. coli and its proteins were more heat sensitive than those of E. coli, simple incubation at 60 degrees C could inactivate most enzymes but the nanA protein. Subsequent column works for purification, then, became simple and rapid.
No preview · Article · Apr 1997 · Analytical Biochemistry
[Show abstract][Hide abstract]ABSTRACT: Various plasmids harboring the truncated DNA polymerase gene (polA) from Thermus thermophilus HB8 (Tth polymerase) were constructed. The most thermostable TthΔNF2 polymerase [the gene product of polAΔNF2, which lacked a 751-bp region (region flanked by initiation codon and FspI 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 TthΔNF2 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 TthΔNF2 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 TthΔNF2 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 TthΔNF2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.
No preview · Article · Dec 1996 · Journal of Fermentation and Bioengineering
[Show abstract][Hide abstract]ABSTRACT: N-Terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) lacking 5'-3' exonuclease activity was used for DNA sequencing and polymerase chain reaction (PCR). In contrast to the high background of the sequencing ladder observed with the wild-type Tth polymerase, delta Tth polymerase gave readable sequencing patterns which extend up to more than 500 bases from the primer site on cycle sequencing and automated sequencing. The delta Tth polymerase was used for the standard and mutagenic PCR, and net amplification of the DNA and the mutations accumulated during PCR were analyzed. Under mutagenic PCR, the mutation rates were 7.0 x 10(-4) (Tth) and 8.3 x 10(-4) (delta Tth) per nucleotide per cycle of amplification, which were 4-9 times higher than the rates under standard PCR.
[Show abstract][Hide abstract]ABSTRACT: The polymerase chain reaction (PCR) has become an important method in molecular biology, and Tag DNA polymerase is the most widely used enzyme. However, Taq DNA polymerase has no 3'-5' exonuclease activity so it cannot proof-read misincorporated nucleotides. Although a variety of thermostable DNA polymerases with higher fidelity and thermostability have been identified, use of these DNA polymerases has been fairly limited due to their lower extension rate and processivity. We have isolated me hyperthermophilic archeon Pyrococcus sp. KOD1 from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan, previously reported. In this study we have cloned the gene for and characterized the DNA polymerase (KOD DNA polymerase) from mis strain. KOD DNA polymerase resembles Pfu DNA polymerase in molecular weight, thermostability , and fidelity. However, its processivity (above 300 nucleotides) is higher than that afP/u DNA polymerase (below 20 nucleotides). Furthermore, its extension rate (100-130 nucleotides/sec.) is about 5 times higher than that of Pfu DNA polymerase (approx. 25 nucleotides/sec.). These properties of the KOD DNA polymerase make it possible to perform fast and accurate PCR. .
[Show abstract][Hide abstract]ABSTRACT: We synthesized a set of four biotinylated dideoxynucleoside triphosphates (biotin-9-ddNTPs) and optimized the reaction conditions for non-radioactive cycle sequencing using modified Tth DNA polymerase (delta Tth) and a chemiluminescent detection system. The resulting sequencing ladders showed lower background compared to those with the conventional non-radioactive sequencing method which uses 5'-biotinylated primers, especially when PCR products were analysed. With our method, DNA sequences can be determined at any primer positions without preparing 5'-biotinylated primers for dideoxy chain-termination.