Development of hybrid biomicroparticles: cellulose exposing functionalized fusion proteins.

BACKGROUND: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins. RESULTS: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology. Such CBD fusions can be efficiently attached to micro-/nanocellulose to form functional, hybrid bionanoparticles. Microcellulose (MCC) particles were generated by a novel approach to enzymatic hydrolysis using Aspergillus sp. cellulase. The interaction between the constructs components - MCC, CBD and fused concatemeric proteins - was evaluated. Obtaining of hybrid biomicroparticles of a natural cellulose biocarrier with proteins with therapeutic properties, fused with CBD, was confirmed. Further, biological tests on the hybrid bioMCC particles confirmed the lack of their cytotoxicity on 46BR.1 N fibroblasts and human adipose derived stem cells (ASCs). The XTT analysis showed a slight inhibition of the proliferation of 46BR.1 N fibroblasts and ACSs cells stimulated with the hybrid biomicroparticles. However, in both cases no changes in the morphology of the examined cells after incubation with the hybrid biomicroparticles' MCC were detected. CONCLUSIONS: Microcellulose display with recombinant proteins involves utilizing cellulose, a natural polymer found in plants, as a platform for presenting or displaying proteins. This approach harnesses the structural properties of cellulose to express or exhibit various recombinant proteins on its surface. It offers a novel method for protein expression, presentation, or immobilization, enabling various applications in biotechnology, biomedicine, and other fields. Microcellulose shows promise in biomedical fields for wound healing materials, drug delivery systems, tissue engineering scaffolds, and as a component in bio-sensors due to its biocompatibility and structural properties.

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

Bacteriophages of Thermophilic 'Bacillus Group' Bacteria-A Systematic Review, 2023 Update.

Bacteriophages associated with thermophiles are gaining increased attention due to their pivotal roles in various biogeochemical and ecological processes, as well as their applications in biotechnology and bionanotechnology. Although thermophages are not suitable for controlling bacterial infections in humans or animals, their individual components, such as enzymes and capsid proteins, can be employed in molecular biology and significantly contribute to the enhancement of human and animal health. Despite their significance, thermophages still remain underrepresented in the known prokaryotic virosphere, primarily due to limited in-depth investigations. However, due to their unique properties, thermophages are currently attracting increasing interest, as evidenced by several newly discovered phages belonging to this group. This review offers an updated compilation of thermophages characterized to date, focusing on species infecting the thermophilic bacilli. Moreover, it presents experimental findings, including novel proteomic data (39 proteins) concerning the model TP-84 bacteriophage, along with the first announcement of 6 recently discovered thermophages infecting Geobacillus thermodenitrificans: PK5.2, PK2.1, NIIg10.1, NIIg2.1, NIIg2.2, and NIIg2.3. This review serves as an update to our previous publication in 2021.

Modified Peptide Molecules As Potential Modulators of Shelterin Protein Functions; TRF1.

In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown in vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins.

BACKGROUND: In spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host's capsule. RESULTS: The TP84_26 'capsule depolymerase' (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC-MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion. CONCLUSIONS: The novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site.

BACKGROUND: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. RESULTS: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. CONCLUSION: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

Boosting toxic protein biosynthesis: transient in vivo inactivation of engineered bacterial alkaline phosphatase.

BACKGROUND: The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts' metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host's viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli (E. coli) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors' background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation. RESULTS: We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal His6-tag, cloned and expressed in E. coli in a PBAD promoter expression vector. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn2+, Mg2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3'-protruding). CONCLUSIONS: The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change.

Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4-8 bp long, with very few exceptions cleaving DNA more frequently. TsoI is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus-family exhibit 'affinity star' activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue-sinefungin (SIN). Here, we define a novel type of inherently built-in 'star' activity, exemplified by TsoI. The TsoI 'star' activity cannot be described under the definition of the classic 'star' activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The TsoI SCS comprises several degenerated variants of the cognate site. Although the efficiency of TsoI SCS cleavage is lower in comparison to the cognate TsoI recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The TsoI/SAC REase may serve as a novel tool for DNA manipulation.

The third restriction-modification system from Thermus aquaticus YT-1: solving the riddle of two TaqII specificities.

Two restriction-modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5'-TCGA-3'. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different asymmetric sequences: 5'-GACCGA-3' and 5'-CACCCA-3'. We previously cloned the taqIIRM gene, purified the recombinant protein from Escherichia coli, and showed that TaqII recognizes the 5'-GACCGA-3' sequence only. Here, we report the discovery, isolation, and characterization of TaqIII, the third R-M system from T. aquaticus YT-1. TaqIII is a 1101-aa Type IIC/IIL enzyme and recognizes the 5'-CACCCA-3' sequence previously attributed to TaqII. The cleavage site is 11/9 nucleotides downstream of the A residue. The enzyme exhibits striking biochemical similarity to TaqII. The 93% identity between their aa sequences suggests that they have a common evolutionary origin. The genes are located on two separate plasmids, and are probably paralogs or pseudoparalogs. Putative positions and aa that specify DNA recognition were identified and recognition motifs for 6 uncharacterized Thermus-family enzymes were predicted.

Randomized DNA libraries construction tool: a new 3-bp 'frequent cutter' TthHB27I/sinefungin endonuclease with chemically-induced specificity.

BACKGROUND: Acoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~ 3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN. RESULTS: In this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5'-CAARCA-3' is converted into a combined 3.2-3.0-bp 'site' or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed. CONCLUSIONS: In the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA.

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site.

The aim of this study was to improve a useful molecular tool-TaqII restriction endonuclease-methyltransferase-by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue-TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.

Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection.

DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid.

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) PR 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.

Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N473A variant in the NPPY motif.

We reported previously that TspGWI, a prototype enzyme of a new Thermus sp. family of restriction endonucleases-methyltransferases (REases-MTases), undergoes the novel phenomenon of sinefungin (SIN)-caused specificity transition. Here we investigated mutant TspGWI N473A, containing a single amino acid (aa) substitution in the NPPY motif of the MTase. Even though the aa substitution is located within the MTase polypeptide segment, DNA cleavage and modification are almost completely abolished, indicating that the REase and MTase are intertwined. Remarkably, the TspGWI N473A REase functionality can be completely reconstituted by the addition of SIN. We hypothesize that SIN binds specifically to the enzyme and restores the DNA cleavage-competent protein tertiary structure. This indicates the significant role of allosteric effectors in DNA cleavage in Thermus sp. enzymes. This is the first case of REase mutation suppression by an S-adenosylmethionine (SAM) cofactor analogue. Moreover, the TspGWI N473A clone strongly affects E. coli division control, acting as a 'selfish gene'. The mutant lacks the competing MTase activity and therefore might be useful for applications in DNA manipulation. Here we present a case study of a novel strategy for REase activity/specificity alteration by a single aa substitution, based on the bioinformatic analysis of active motif locations, combining (a) aa sequence engineering (b) the alteration of protein enzymatic properties, and (c) the use of cofactor-analogue cleavage reconstitution and stimulation.

A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries.

BACKGROUND: Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases). RESULTS: We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) - an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5'-GACCGA-3' [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation. CONCLUSIONS: In the presence of SIN/DMSO, TaqII REase is transformed from cleaving every 4096 bp on average to cleaving every 58 bp. TaqII SIN/DMSO thus extends the palette of available REase prototype specificities. This phenomenon, employed under partial digestion conditions, was applied to quasi-random DNA fragmentation. Further applications include high sensitivity probe generation and metagenomic DNA amplification.

Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus.

BACKGROUND: In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI. RESULTS: TsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined. CONCLUSIONS: Previously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism's thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.

A Method for Rapid Polyethyleneimine-Based Purification of Bacteriophage-Expressed Proteins from Diluted Crude Lysates, Exemplified by Thermostable TP-84 Depolymerase.

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA-protein complexes or PEI-acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme's properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion.

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

BACKGROUND: We previously defined a family of restriction endonucleases (REases) from Thermus 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. RESULTS: 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 Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases. CONCLUSIONS: 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.

A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus.

DNA purification methods are indispensable tools of molecular biology, used for many decades. Nevertheless, for certain specialized applications, the currently employed techniques are not sufficiently effective. While examining a number of the existing methods to purify the genomic DNA of the thermophilic bacteriophage TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus), we have found out that the obtained DNA is contaminated with trace amounts of infectious TP-84 particles. This was detrimental for the bacteriophage genetic manipulation purposes, as finding the recombinant TP-84 clones was essentially impossible due to the appearance of a high background of native bacteriophage plaques. Thus, we have developed a method, which enables the fast and efficient isolation of a bacteriophage genomic DNA from concentrated phage preparations, obtained using CsCl gradient ultracentrifugation, without the need to remove concentrated CsCl solutions. The method employs silica columns and mini-scale isolation of microgram amounts of high quality DNA. It is universal-the silica mini-columns from various manufacturers can be used to conduct the procedure. The purified DNA, free from infectious bacteriophage particles, is ready for further manipulations. This is particularly important for such thermophilic bacteriophages that may partially survive standard isolation procedures and contaminate the final DNA product.