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.

Applications of the phage display technology in molecular biology, biotechnology and medicine.

The phage display technology is based on the presentation of peptide sequences on the surface of virions of bacteriophages. Its development led to creation of sophisticated systems based on the possibility of the presentation of a huge variability of peptides, attached to one of proteins of bacteriophage capsids. The use of such systems allowed for achieving enormous advantages in the processes of selection of bioactive molecules. In fact, the phage display technology has been employed in numerous fields of biotechnology, as diverse as immunological and biomedical applications (in both diagnostics and therapy), the formation of novel materials, and many others. In this paper, contrary to many other review articles which were focussed on either specific display systems or the use of phage display in selected fields, we present a comprehensive overview of various possibilities of applications of this technology. We discuss an usefulness of the phage display technology in various fields of science, medicine and the broad sense of biotechnology. This overview indicates the spread and importance of applications of microbial systems (exemplified by the phage display technology), pointing to the possibility of developing such sophisticated tools when advanced molecular methods are used in microbiological studies, accompanied with understanding of details of structures and functions of microbial entities (bacteriophages in this case).

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.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation.

BACKGROUND: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host. RESULTS: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels. CONCLUSIONS: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

N-Terminally Lipidated Sialorphin Analogs-Synthesis, Molecular Modeling, In Vitro Effect on Enkephalins Degradation by NEP and Treatment of Intestinal Inflammation in Mice.

Pharmacotherapy for inflammatory bowel disease (IBD) is difficult, and some patients do not respond to currently available treatments. Therefore, the discovery of novel anti-IBD agents is imperative. Our aim was the synthesis of lipidated analogs of sialorphin and the in vitro characterization of their effect on the degradation of Met-enkephalin by neutral endopeptidase (NEP). We also investigated in vivo whether the most active inhibitor (peptide VIII) selected in the in vitro studies could be a potential candidate for the treatment of colitis. Peptides were synthesized by the solid-phase method. Molecular modeling technique was used to explain the effect of fatty acid chain length in sialorphin analogs on the ligand-enzyme interactions. The anti-inflammatory effect was evaluated in the dextran sulphate sodium (DSS)-induced model of colitis in mice. Peptide VIII containing stearic acid turned out to be in vitro the strongest inhibitor of NEP. We have also shown that the length of the chain of stearic acid fits the size of the grove of NEP. Peptides VII and VIII exhibited in vivo similar anti-inflammatory activity. Our results suggest that lipidation of sialorphin molecule is a promising direction in the search for NEP inhibitors that protect enkephalins.

Cloning and Characterization of a Thermostable Endolysin of Bacteriophage TP-84 as a Potential Disinfectant and Biofilm-Removing Biological Agent.

The obligatory step in the life cycle of a lytic bacteriophage is the release of its progeny particles from infected bacterial cells. The main barrier to overcome is the cell wall, composed of crosslinked peptidoglycan, which counteracts the pressure prevailing in the cytoplasm and protects the cell against osmotic lysis and mechanical damage. Bacteriophages have developed two strategies leading to the release of progeny particles: the inhibition of peptidoglycan synthesis and enzymatic cleavage by a bacteriophage-coded endolysin. In this study, we cloned and investigated the TP84_28 endolysin of the bacteriophage TP-84, which infects thermophilic Geobacillus stearothermophilus, determined the enzymatic characteristics, and initially evaluated the endolysin application as a non-invasive agent for disinfecting surfaces, including those exposed to high temperatures. Both the native and recombinant TP84_28 endolysins, obtained through the Escherichia coli T7-lac expression system, are highly thermostable and retain trace activity after incubation at 100 °C for 30 min. The proteins exhibit strong bacterial wall digestion activity up to 77.6 °C, decreasing to marginal activity at ambient temperatures. We assayed the lysis of various types of bacteria using TP84_28 endolysins: Gram-positive, Gram-negative, encapsulated, and pathogenic. Significant lytic activity was observed on the thermophilic and mesophilic Gram-positive bacteria and, to a lesser extent, on the thermophilic and mesophilic Gram-negative bacteria. The thermostable TP84_28 endolysin seems to be a promising mild agent for disinfecting surfaces exposed to high temperatures.

Butyrylcholinesterase-Protein Interactions in Human Serum.

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein-protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes' specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke.

Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH2 (R49KKRRQRRR57-amide) and its less basic analogue, PTD4 (Y47ARAAARQARA57-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 µm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH2 adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH2) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.

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.

An alternative for proteinase K-heat-sensitive protease from fungus Onygena corvina for biotechnology: cloning, engineering, expression, characterization and special application for protein sequencing.

BACKGROUND: A neutral, heat-sensitive serine protease (NHSSP) originating from the feather-degrading fungus Onygena corvina (O. corvina) was described and defined as an alkaline serine protease of the subtilisin type S8 family, exhibiting an enzymatic activity at neutral pH. Generally, broad specificity proteases, such as proteinase K or trypsin, have found numerous applications in research and biotechnology. RESULTS: We report the cloning and expression in the yeast PichiaPink™ system, as well as purification, and characterization of the NHSSP. Recombinant, His6-tagged NHSSP was efficiently expressed from an optimized, synthetic gene and purified using a simple protocol based on ammonium sulfate fractionation and hydrophobic interaction chromatography. The enzyme shows atypical C-terminal processing, the coded preproprotein undergoes signal peptide removal and maturation through the clipping of a propeptide section and 10 amino acids (aa) from the C-terminus, including the His6-tag. The deletion variant has been constructed, devoid of the C-terminal ORF segment, thus eliminating the need for C-terminal processing. Both NHSSP variants exhibit very similar enzymatic characteristics. The purified enzymes were characterized to determine the optimal proteolytic conditions. We revealed that the mature NHSSP is reproducibly active over a wide pH range from neutral to mild acidic (pH of 5.0 to 8.5), with an optimum at pH 6.8, and at temperatures of 15 to 50 °C with an optimum at 38-42 °C. Interestingly, we demonstrated that the protease can be fully deactivated by a moderate increase in temperature of about 15 °C from the optimum to over 50 °C. The protease was partially sensitive to serine protease inhibitors, and not inhibited by chelating or reducing agents and detergents. SDS induced autolysis of NHSSP, which points to a high stimulation of its proteolytic activity. CONCLUSIONS: The NHSSP was produced as a recombinant protein with high efficiency. Compared to proteinase K, the most common serine protease used, NHSSP shows an approx. twofold higher specific activity. Protein sequencing can be a valuable technical application for the protease. The protein coverage is significantly higher in comparison to trypsin and reaches about 84-100% for ß-lactoglobulin (BLG), antibody (mAb) light and heavy chains. Furthermore, the option to perform digestions at neutral to slightly acidic pH-values down to pH 5.0 avoids modification of peptides, e.g. due to deamidation.

Imunofan-RDKVYR Peptide-Stimulates Skin Cell Proliferation and Promotes Tissue Repair.

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today's science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name "Imunofan" (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30-40% and 20-50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

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.