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

Abstract 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

Increased levels of antibodies against heat shock proteins in stroke patients

Ischemic stroke is the second leading cause of death worldwide. One of the main risk factors of the ischemic stroke is atherosclerosis which is a chronic inflammatory and immune-mediated disease. Bacterial infections generate specific human antibodies against various antigens, including Hsps. It has been demonstrated that Hsps are selectively overexpressed in the atherosclerotic lesions. The amino acid sequence homology between human and bacterial Hsps may lead to an autoimmune response by immunological cross-reaction. Such immune response against Hsps overexpressed in the blood vessels under stressful conditions may contribute to inflammatory processes and subsequent development of atherosclerosis. In this study we determined the antibody levels against bacterial and human Hsp by ELISA in blood plasma obtained from stroke patients. Using ANOVA we analyzed levels of Hsp-antibodies in control and patient groups and correlate them with several stroke risk factors. The group of stroke patients had elevated levels of anti-Hsp antibodies compared to the control group. We also discovered an antibody level increase in patients that previously underwent another stroke. Our data provide evidence that autoimmunity could underlie formation of atherosclerosis plaque leading to stroke

Comparison of RM.TthHB27I thermozyme production in native wt host <i>T</i>. <i>thermophilus</i> and in <i>E</i>. <i>coli</i> expressing both <i>tthHB27IRM</i> gene variants.

<p>Comparison of RM.TthHB27I thermozyme production in native wt host <i>T</i>. <i>thermophilus</i> and in <i>E</i>. <i>coli</i> expressing both <i>tthHB27IRM</i> gene variants.</p

Biosynthesis and activity of synthetic RM.TthHB27I.

<p>(A) Isolation of synthetic RM.TthHB27I from <i>E</i>. <i>coli</i>. Lane M1, PageRuler™ Unstained Broad Range Protein Ladder; lane M2, Pierce™ Unstained Protein Molecular Weight Marker; lane 1, native wt RM.TthHB27I; lane 2, crude lysate from <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>], grown at 30°C; lane 3, supernatant after PEI treatment; lane 4, supernatant after incubation at 65°C; lane 5, 0–50% AmS fractionation cut; lane 6, DEAE-cellulose chromatography; lane 7, heparin-agarose chromatography; lane 8, Phosphocellulose P11 chromatography. (B) Yields of recombinant wt RM.TthHB27I and synthetic RM.TthHB27I biosynthesis. Recombinant <i>E</i>. <i>coli</i> BL21(DE3) strains were subjected to 3 h induction at OD₆₀₀ = 0.6–0.7 and 30°C. Cells were lysed and protein lysates were analysed by 7.5% SDS-PAGE. Lanes M1, M2 as in (A); lane 1, synthetic RM.TthHB27I; lane 2, crude lysate from induced <i>E</i>. <i>coli</i> [pET21d(+)-wt-<i>tthHB27IRM</i>]; lane 3, crude lysate from induced <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>]. (C) Comparison of the activities of RM.TthHB27I MTase variants <i>in vivo</i>. 0.5 μg of total DNA from <i>T</i>. <i>thermophilus</i> or induced, recombinant <i>E</i>. <i>coli</i> BL21(DE3) strains were digested with 2 units of synthetic RM.TthHB27I in REase buffer+SAM for 1 h at 65°C. Lane 1, untreated <i>T</i>. <i>thermophilus</i> DNA; lane 2, <i>T</i>. <i>thermophilus</i> DNA digested with synthetic RM.TthHB27I; lane 3, untreated <i>E</i>. <i>coli</i> BL21(DE3) [pET21d(+)-wt-<i>tthHB27IRM</i>] DNA; lane 4, as in lane 3, but with synthetic RM.TthHB27I; lane 5, untreated <i>E</i>. <i>coli</i> BL21(DE3) [pET21d(+)-synthetic <i>tthHB27IRM</i>] DNA; lane 6, as in lane 5, but with synthetic RM.TthHB27I; lane M, GeneRuler 1 kb DNA Ladder.</p

Predicted secondary structure of the first 200 nt of <i>tthHB27IRM</i> mRNA generated by Mfold Web Server [34, 35].

<p>(A) Predicted structure of initial recombinant wt <i>tthHB27IRM</i> mRNA fragment before codon optimization (revised free energy: dG = -60.40 kcal/mol). (B) Structure of initial synthetic <i>tthHB27IRM</i> mRNA fragment after codon optimization (revised free energy: dG = -68.04 kcal/mol).</p

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from <i>Thermus thermophilus</i> (RM.TthHB27I) cloned in <i>Escherichia coli</i> is critically affected by the codon composition of the synthetic gene

<div><p>Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes’ expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. <i>Thermus thermophilus</i> HB27 (<i>T</i>. <i>thermophilus</i>) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into <i>E</i>. <i>coli</i>, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic <i>tthHB27IRM</i> gene, using a modified ‘codon randomization’ strategy. Codons with a high GC content and of low occurrence in <i>E</i>. <i>coli</i> were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant <i>tthHB27IRM</i> gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from <i>T</i>. <i>thermophilus</i>. Thus, we have developed an efficient purification protocol using the synthetic <i>tthHB27IRM</i> gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.</p></div

Expression of synthetic <i>tthHB27IRM</i> gene as a function of temperature.

<p>(A) Kinetics of <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>] bacterial cultures growth and synthetic RM.TthHB27I protein expression at 30°C. The recombinant <i>E</i>. <i>coli</i> BL21(DE3) cultures were cultivated in TB media at 30°C with vigorous aeration. After induction (OD₆₀₀ = 0.6–0.7) with IPTG cultures were grown for 6 h. Culture samples were taken at 1 h intervals and subjected to spectrophotometric analysis and SDS-PAGE. Cells were lysed and lysates were analysed using 7.5% SDS-PAGE. Lane M1, PageRuler™ Unstained Broad Range Protein Ladder (Thermo Fisher Scientific/Fermentas); lane M2, Pierce™ Unstained Protein Molecular Weight Marker (Thermo Fisher Scientific/Fermentas); lane 1, synthetic RM.TthHB27I protein; lane 2, separate control culture <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>] cultivated at 30°C, before induction (OD₆₀₀ = 0.6); lane 3, control culture—6 h after induction; lane 4, <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>] experimental culture, before induction (OD₆₀₀ = 0.6); lane 5, 1 h after induction; lane 6, 2 h; lane 7, 3 h; lane 8, 4 h; lane 9, 5 h; lane 10, 6 h. (B) Experiment conducted as in (A), but at 37°C. (C) Experiment conducted as in (A), but at 42°C. (D) Experiment conducted as in (A), but at 46°C.</p

Additional file 1: of Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

PCR fragment DNA substrates nucleotide sequences. (A) 1789 bp PCR fragment DNA, containing two convergent (→←) TthHB27I canonical sites. Recognition sequence is indicated in bold and underlined. Arrows indicate the cleavage points. Restriction fragments lenght: 311, 602 and 872 bp. (B) 1850 bp PCR fragment DNA without TthHB27I site. (TIF 673 kb

Additional file 2: of Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

TthHB27I specificity change in the presence of SAM and DMSO. (PDF 26 kb