Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

<p>Abstract</p> <p>Background</p> <p>Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a <it>Thermus </it>sp. enzyme family, which belongs to type II restriction endonucleases (REases), however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II.</p> <p>Results</p> <p>Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the <it>tspgwi </it>gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the <it>Thermus </it>sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase) and methyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolated from <it>Thermus </it>sp.) are dependent on the presence of divalent cations.</p> <p>Conclusion</p> <p>TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI are autonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appears to be a streamlined 'half' of a Type I enzyme.</p

DNA-FACE™ - An <em>Escherichia coli</em>-based DNA Amplification-Expression Technology for Automatic Assembly of Concatemeric ORFs and Proteins

DNA-FACE™ (DNA Fragment Amplification & Concatemeric Expressed Nucleic Acids and Proteins) is a universal biotechnological platform, developed as Escherichia coli (E. coli) system. It is based on the ordered, head-to-tail directional ligation of the amplified DNA fragments. The technology enables the construction of targeted biomolecules - genetically programmed, concatemeric DNA, RNA, and proteins, designed to fit a particular task. The constructed, “artificial” (never seen in Nature) tandem repeat macromolecules, with specialized functions, may contain up to 500 copies of monomeric units. The technology greatly exceeds the current capabilities of chemical gene synthesis. The vector-enzymatic DNA fragment amplification assembles the DNA segments, forming continuous Open Reading Frames (ORFs). The obtained ORFs are ready for high-level expression in E. coli without a need for subcloning. The presented method has potential applications in pharmaceutical industry and tissue engineering, including vaccines, biological drugs, drug delivery systems, mass-production of peptide-derived biomaterials, industrial and environmental processes. The technology has been patented worldwide and used successfully in the construction of anti-HBV vaccines, pro-regenerative biological drugs and, recently, the anti-SARS-CoV-2 vaccine. The anti-SARS-CoV-2 vaccine, developed using the DNA-FACE™ technology, is nontoxic and induces strong immunological response to recombinant human spike and nucleocapsid proteins, as shown in animal studies

Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

<p>Abstract</p> <p>Background</p> <p>The TaqII enzyme is a member of the <it>Thermus </it>sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various <it>Thermus </it>sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III.</p> <p>Results</p> <p>Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC).</p> <p>Conclusions</p> <p>The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from <it>Thermus aquaticus </it>has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of the Fidelity Index will be helpful for DNA manipulation with enzymes only partially cleaving DNA.</p

Modified ‘one amino acid-one codon’ engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase

BACKGROUND: An industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host’s viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host’s coding plasmid replication. TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM). RESULTS: We describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) P( R ) promoter. Codon usage based on a modified ‘one amino acid–one codon’ strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields ‘codon randomization’ strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high ‘toxicity’ of the REase-MTase protein. CONCLUSIONS: Recombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified ‘one amino acid–one codon’ method tuned for thermophile-coded genes was applied to obtain overexpression of the ‘toxic’ taqIIRM gene. The method appears suited for industrial production of thermostable ‘toxic’ enzymes in E. coli. This novel variant of the method biased toward increasing a gene’s AT content may provide economic benefits for industrial applications

Microbiological characterization of psychro-mezo-thermophilic endospore-producing Bacillus species isolated from industrial probiotics particles

Probiotics are either bacteria which naturally and steadily reside in the human gastrointestinal tract (GIT), such as certain Lactobacillus sp., or are bimodal, i.e. capable of proliferation both in GIT, as well as in the external environment, these include certain Bacillus sp. In this report we characterize a mixture of Bacillus species present in widely used commercial preparations, present in lyophilized particles. Four endospore-producing species were detected through MALDI TOF mass spectrometry and microbiological analyses: Bacillus mojavensis, Bacillus vallismortis, Bacillus pumilus and Bacillus subtilis. They exhibit an exceptionally wide range of growth temperature: from 20°C to 58oC, thus they are environmentally multi-modal and cover areas occupied both by psychrophiles, mesophiles and thermophiles. Thus, they are exceedingly adaptive to different environments and able to proliferate in highly diverse niches, including the human GIT. Considering that all of the four characterized species have similar characteristics, including endospore production and growth in a wide range of pH, which allows them to survive in transiently low pH during GIT passage, as well as their widespread occurrence in the environment, it is very likely that they have evolved along with mammals as their natural, transient or permanent, GIT inhabitants, though they are not limited to this niche. Acknowledgments: the project was supported GRUPA INCO S.A., ul. Wspolna 25, 00-519 Warsaw, Poland, NCBiR grant no POIG.01.04.00-02-181/13 and by University of Gdansk task funds no. DS 530-8645-D509-15

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

<p>Abstract</p> <p>Background</p> <p>We previously defined a family of restriction endonucleases (REases) from <it>Thermus </it>sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.</p> <p>Results</p> <p>TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the <it>Thermus </it>sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.</p> <p>Conclusions</p> <p>TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.</p

Construction of bionanoparticles with the use of a recombinant DNA vector-enzymatic system, containing artificial poliepitopic proteins, for the delivery of new generation vaccines

DNA/RNA amplification technologies, such as the Polymerase Chain Reaction have revolutionized modern biology, medical diagnostics and forensic analyses, among others. A number of alternative nucleic acids amplification methods have been developed, tailored to specific applications. Here we present a refined version of a DNA fragment amplification technology, which enables the construction of ordered concatemers in a head-to-tail-orientation. A very high number of DNA segments, at least 500 copies, can be consecutively linked. Other key features include: (i) the application of a dedicated vector-enzymatic system, including selected subtype IIS restriction endonucleases, which has been designed to automatically generate long Open Reading Frames and (ii) an amplification-expression vector with a built-in strong transcription promoter along with optimal translation initiation signals, which allow for a high level of expression of the constructed artificial poliepitopic protein. This highly advanced technology makes it possible to obtain ordered polymers of monomeric, synthetic or natural, DNA far beyond the capabilities of current chemical synthesis methods. The constructed poliepitopic proteins are further used for construction of several types of nanoparticles, including inclusion bodies and bacteriophages, containing multiple genetic fusion with poliepitopic proteins.The technology offers significant advances in a number of scientific, industrial and medical applications, including new vaccines and tissue pro-regenerative methods. The technology is protected by an international patent application and is available for licensing. Acknowledgments: project was supported by National Center for Research and Development, Warsaw, Poland, grant no STRATEGMED1/235077/9/NCBR/2014 and POIG.01.04.00-22-140/12; Jagiellonian Center for Innovation, Krakow, Poland; SATUS VC, Warsaw, Poland and BioVentures Institute Ltd, Poznan, Poland

The OGLE Collection of Variable Stars. One Thousand Heartbeat Stars in the Galactic Bulge and Magellanic Clouds

We present a collection of 991 heartbeat star (HBS) candidates found in the Optical Gravitational Lensing Experiment (OGLE) project data archive. We discuss the selection process of the HBS candidates and the structure of the catalog itself. It consists of 512 stars located toward the Galactic bulge (GB), 439 stars located in the Large Magellanic Cloud (LMC), and 40 in the Small Magellanic Cloud (SMC). The collection contains two large groups of HBSs with different physical properties. The main distinction between the two groups is the evolutionary status of the primary star. The first group of about 100 systems contains a hot main-sequence (MS) or a Hertzsprung-gap primary star, while the second group of about 900 systems includes a red giant (RG). For each star, we provide two-decade-long time-series photometry, in the Cousins $I$- and Johnson $V$-band filters, obtained by the OGLE project. We also present basic observational information as well as orbital parameters derived from the light curve modeling.Comment: 15 pages, 10 figures, 5 tables, submitted to AAS Journals. The full machine-readable tables and links to the catalog will be available after the acceptance of the pape

The Araucaria Project: A study of the classical Cepheid in the eclipsing binary system OGLE LMC562.05.9009 in the Large Magellanic Cloud

We present a detailed study of the classical Cepheid in the double-lined, highly eccentric eclipsing binary system OGLE-LMC562.05.9009. The Cepheid is a fundamental mode pulsator with a period of 2.988 days. The orbital period of the system is 1550 days. Using spectroscopic data from three 4-8-m telescopes and photometry spanning 22 years, we were able to derive the dynamical masses and radii of both stars with exquisite accuracy. Both stars in the system are very similar in mass, radius and color, but the companion is a stable, non-pulsating star. The Cepheid is slightly more massive and bigger (M_1 = 3.70 +/- 0.03M_sun, R_1 = 28.6 +/- 0.2R_sun) than its companion (M_2 = 3.60 +/- 0.03M_sun, R_2 = 26.6 +/- 0.2R_sun). Within the observational uncertainties both stars have the same effective temperature of 6030 +/- 150K. Evolutionary tracks place both stars inside the classical Cepheid instability strip, but it is likely that future improved temperature estimates will move the stable giant companion just beyond the red edge of the instability strip. Within current observational and theoretical uncertainties, both stars fit on a 205 Myr isochrone arguing for their common age. From our model, we determine a value of the projection factor of p = 1.37 +/- 0.07 for the Cepheid in the OGLE-LMC562.05.9009 system. This is the second Cepheid for which we could measure its p-factor with high precision directly from the analysis of an eclipsing binary system, which represents an important contribution towards a better calibration of Baade-Wesselink methods of distance determination for Cepheids.Comment: Accepted to be published in Ap