Recombinant Nuclease Research Articles

Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.

2'-deoxynucleoside 5'-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.

Construction of a Z-DNA-Specific Recombinant Nuclease Zαα-FOK for Conformation Studies.

Development of FokI-based engineered nucleases has been a platform technology that enables creation of novel sequence-specific nucleases as well as structure-specific nucleases. Z-DNA-specific nucleases have been constructed by fusing a Z-DNA-binding domain to the nuclease domain of FokI (FN). In particular, Zαα, an engineered Z-DNA-binding domain with a high affinity, is an ideal fusion partner to generate a highly efficient Z-DNA-specific cutter. Here, we describe construction, expression, and purification of Zαα-FOK (Zαα-FN) nuclease in detail. In addition, Z-DNA-specific cleavage is demonstrated by the use of Zαα-FOK.

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair.

Simple SummaryApproaches to manipulate the genome of an organism, both selectively and accurately, are powerful techniques that can influence research practice, with extensive application to agriculture and medicine, including the ability to impact disease risk or onset. In this review article, we highlight the advances, made over several decades, on the procedures and capacities to facilitate genome editing, manifest with the discovery, characterization, and optimization of the mechanism for processing of clustered regularly interspaced short palindromic repeat sequences (CRISPR). The editing molecules in the CRISPR gene modification system behave as molecular scissors, cutting DNA at specific genetic locations. First identified as a natural defense mechanism in bacteria, the CRISPR system has now been extensively modified for use in almost all mammalian cells. In describing each CRISPR mechanistic class, we acknowledge the differences and positive attributes each class has to offer to support editing that allows the creation of gene knockouts, knock-ins, gene tagging, insertions, deletions, and point mutations. Further, we discuss how these editing strategies have shaped the field of DNA repair. Specifically, we provide examples of the utility of CRISPR approaches in furthering our understanding of two of the major DNA repair pathways, namely mismatch repair and base excision repair.The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

In vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.

We established an in vitro clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame, promoter, terminator, and directional repeats (DRs) flanking sequences. It was cotransformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild-type strain, were able to grow on the media containing acetamide as the sole nitrogen source and cesium chloride. Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop-out of the amdS expression cassette by recombining the flanking DRs. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter-selection in gene editing of A. niger. The proposed in vitro CRISPR/Cas9 method offers a powerful tool for marker-free genetic manipulation of filamentous fungi industrial-specific strains.

Abstract 6137: Development of ARID1A-deficient hepatocellular carcinoma in transgenic pigs

Abstract Hepatocellular carcinoma (HCC) is an aggressive disease with poor response to treatment at advanced stages. Frameshift and nonsense mutations in ARID1A, a subunit of the SWI/SNF chromatin-remodeling complex, are among the most frequent genetic alterations in human HCC. Hence, it is necessary to develop animal models for preclinical assessment of targeted therapies for HCC with ARID1A deficiency. Due to the remarkable physiologic, genetic, and size similarities between pigs and humans, porcine models are excellent candidates for translational HCC studies. Here, we generated a porcine HCC model with ARID1A deficiency using the Oncopig, a transgenic pig that harbors heterozygous Cre recombinase-inducible TP53R167H and KRASG12D transgenes. Porcine HCC cells were developed by transduction of hepatocytes isolated from four Oncopigs with adenoviral vector encoding Cre recombinase. TP53R167H and KRASG12D expression in the developed HCC cell lines was confirmed by RT-PCR, and the hepatocyte origin of the cells was validated by positive arginase-1 staining. To disrupt ARID1A expression, Oncopig HCC cell lines were transfected with recombinant Cas9 nuclease complexed with a gRNA targeting ARID1A. This resulted in >85% ARID1A editing in each of the four cell lines as determined by targeted Illumina sequencing. Single cell clones were isolated and screened for ARID1A mutations. The functional effect of ARID1A deficiency was evaluated in ARID1A-edited cell pools and ARID1A knockout single cell clones. Consistent with its reported tumor suppressor role, deficiency of ARID1A increased Oncopig HCC cell proliferation as measured by MTS assays. Following in vitro characterization, ARID1A-deficient Oncopig HCC cells were injected autologously into subcutaneous sites in Oncopig flanks, resulting in development of masses measuring 1-2 cm within two weeks. These subcutaneous masses were excised and fragmented, and 2 - 3 fragments were engrafted into the liver of the same animal by ultrasound-guided injection. Development of intrahepatic masses was detected by ultrasound imaging as well as CT scans. Following euthanasia of the Oncopigs, the intrahepatic masses were collected for histological and genomic analyses. Subcutaneous and intrahepatic masses were histologically characterized as HCC, and CRISPR-Cas9 mediated ARID1A deficiency was confirmed by targeted Illumina sequencing. To conclude, we have developed a novel large animal model of ARID1A-deficient HCC and demonstrated the feasibility of generating genetically-tailored HCC tumors in Oncopigs. These translational HCC models are promising tools for testing precision medicine approaches and investigating the contribution of clinically-relevant driver mutations on treatment susceptibility. Citation Format: Lobna Elkhadragy, William M. Totura, Kimia Dasteh Goli, Lawrence B. Schook, Ron C. Gaba, Kyle M. Schachtschneider. Development of ARID1A-deficient hepatocellular carcinoma in transgenic pigs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6137.

Analysis of DNA Processing Enzyme FEN1 and Its Regulation by Protein Lysine Acetylation.

Cellular proteins are modified by lysine acetylation wherein an acetyltransferase transfers an acetyl group from acetyl co enzyme A onto the e-amino group of lysine residues. This modification is extremely dynamic and can be reversed by a deacetylase that removes the acetyl group. Addition of acetyl group to the lysine residue neutralizes its positive charge, thereby functioning as a molecular switch in regulating the enzymatic functions of the protein, its stability, and it cellular localization. Since this modification is extremely dynamic within the cell, biochemical studies characterizing changes in protein function are imperative to understand how this modification alters protein function in a specific cellular pathway. This unit describes in detail expression and purification of a recombinant nuclease and acetyltransferase, in vitro acetylation of the recombinant protein and biochemical assays to study the changes in enzymatic activity of the in vitro acetylated nuclease.

Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35°C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway.

CRISPR/Cas9-Mediated Genome Editing of Epigenetic Factors for Cancer Therapy.

Advances in engineered recombinant nuclease have provided facile and reliable methods for genome editing. Especially with the development of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) system, the discovery of various versions of Cas9 proteins and delivery carriers, it is now practicable to introduce desired mutations into the genome, to correct disease-related mutations, and to activate or suppress genes of interest. Epigenetic regulators are often disturbed in cancer cells and are essential for the transformation of normal to cancerous cells. Tumor-related epigenetic alterations or epigenetic factor mutations play a major part during the various steps of carcinogenesis and affect a variety of cancer-related genes and a wide range of cancerous phenotypes. Therefore, epigenetic regulatory enzymes might be candidate targets for cancer therapy. In this review, we discuss prospects of CRISPR/Cas9-based genome editing in targeting epigenetics for cancer gene therapy.

Ribonucleases, Nucleases and Antiangiogenins in Antiproliferative Activities

The animal ribonucleases and plant nucleases are selective cytotoxic enzymes with several biological activities inclusively also with antitumorous effects. The first attention from animal researches is dated to 1938 year, meanwhile plant enzyme investigators awoke their attention in last time. The bovine pancreatic ribonuclease, after the first structurally studies, allured very soon the peoples with functionally interests. The results of studies directed to antitumor effect were firstly very weak. The latter isolated bovine seminal ribonuclease was more effective from this aspect. Also human pancreatic ribonuclease stimulated interest about its antitumor activity what was realized by its dimerization and other molecular studies. Ribonuclease inhibitors have been very limited substances in the cytotoxicity of antitumor effectivity. On the other side a conjugation of cytostatic enzymes with polymers enhanced the antitumor activity of them and lowered their toxicity. Plants as producers of new engineered pharmaceuticals are becoming a very attractive tool to improve human health. In last years the main attention from the plant antitumor effects begun to devote to plant ribonucleases and mainly nucleases I. These nucleases, major plant sugar non-specific endonucleases form rather heterogenous group of catalytically related enzymes. An important property that suggests intimate heterogeneity within the nuclease group is the specificity towards secondary structures of nucleic acids and the ability to cleave different homopolymers. Angiogenin, as RNase initiates vascularization of tumors and subsequent tumor growth. The new substances against angiogenin and its process angiogenesis are now in the centrum of antitumor-effort. The combination of these three main substances – ribonuclease, nucleases and antiangiogenins seems to be perspective for further studies. The preliminary results with some of them (bovine pancreatic RNase A, or bovine seminal BS-RNase with tomato recombinant nuclease and antiangiogenesis neamine, hyaluronidase, and melatonin is our first step. Keywords: Angiogenin, anticancer, antiproliferative, cytotoxic, effect, human, nuclease, plant, ribonuclease, cell, therapy, traumatic, brain, injury, adult, pediatric, primary, secondary, angiogenesis, neurogenesis, functional, recovery, endogenuis, exogenius, factors, dedifferentiation, pluripotent, multipotent, Proliferation, Survival, Precursor cells

Plant Ribonucleases and Nucleases as Antiproliferative Agens Targeting Human Tumors Growing in Mice

The antiproliferative and antitumor effect of leaf ribonuclease was tested in vitro on the human ML-2 tumor cell line and in vivo on athymic nude mice bearing human melanoma tumors. The antiproliferative activity of this plant ribonuclease in vitro studies was negligible. In the experiments in vivo a significant decrease of the tumor size, however was observed. From nucleases the mung bean nuclease (PhA) was studied first from nucleases. The antitumor effect of this enzyme on ML2 human tumor cell line was almost non-effective. However, significant antitumor activity was detected on human melanoma tumors in vivo. The antitumor effect of black pine pollen nuclease (PN) tested in vitro was also negligible. On the other side, in the experiments in vivo a significant decrease of the human melanoma tumor size was observed too. Recombinant plant nucleases of tomato (TBN1) and hop (HBN1) (submitted to patenting under no. PV 2008-384;Z7585) were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant both nucleases were as effective against human melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about ten times lower concentrations compared to the use of bovine seminal RNase (BS-RNase).

Antitumor Effects and Cytotoxicity of Recombinant Plant Nucleases

Recombinant plant nucleases R-TBN1 and R-HBN1 were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant nucleases were as effective against melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about 10 times lower concentrations compared to the use of bovine seminal RNase (BS-RNase). Because the recombinant nucleases R-HBN1 and R-TBN1 share only 67.4% amino acid identity and showed only partial immunochemical cross-reactivity, their similar anticancerogenic effects can be mainly explained by their catalytical similarity. Both recombinant nucleases showed lower degree of aspermatogenesis compared to BS-RNAse and PN nuclease. Unlike BS-RNase, aspermatogenesis induced by both recombinant nucleases could not be prevented by the homologous antibody complexes. Owing to relatively low cytotoxicity on the one hand, and high efficiency at low protein levels on the other, recombinant plant nucleases R-HBN1 and R-TBN1 appear to be stable biochemical agents that can be targeted as potential antitumor cytostatics.

High-Throughput Identification of Mutations Using a Combination of CEL I Fragmentation and SAGE Technology

A new method to detect mutations based on the serial analysis of gene expression (SAGE) technique, ligation-mediated (LM) PCR, and recombinant nuclease CEL I named LM-SAGE assay is reported in the present study. Mismatched DNA heteroduplexes formed from wild-type and mutant DNA are fragmented with CEL I nuclease at the mutant site to produce a double-strand fragment with an overhanging base at the 3'-end. The fragment is ligated to a linker, and digested with MmeI and then ligated to another linker. PCR is performed to amplify the ligation products, and NlaIII is used to release 17-bp tags containing mutation sites followed by purification, concatemerization, cloning, and sequencing. The locations of mutations can be identified from the homology analysis of tags. This new LM-SAGE assay can detect both known and unknown mutations with a sensitivity of 1:50 (mutant:wild-type DNA ratio) in 2.4 x 10(6) copies starting DNA sample. Our results show that this method could be used as a potentially high-throughput assay for mutation detection, particularly for the discovery of unknown mutations in genomic DNA.

Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection

BackgroundThe detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria.ResultsWe describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites.ConclusionThe insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.

Recovery of Nuclease Produced by Lactococcus Lactis Using Expanded Bed Ion Exchange Chromatography

Expanded bed-ionic exchange chromatography (EB-IEC) was used for the recovery and purification of recombinant staphylococcal nuclease secreted by Lactococcus lactis. At the end of the fermentation process, the nuclease activity reached 39 U ml(-1). The EB-IEC performances were firstly evaluated with clarified culture broth. The isocratic elution with 0.5 M NaCl led to approximately 80% of nuclease recovery. Proceeding with 3-fold bed expansion resulted in a reduction of the resin capacity by a factor of 32% compared to the process in a packed bed configuration. Simplification of the early purification steps was reached by loading immediately the unclarified culture broth previously diluted to reduce conductivity. Presence of Cells did not affect the chromatography performances resulting in 55-fold purification with the same yield.

Protein Stabilization by Osmolytes from Hyperthermophiles: EFFECT OF MANNOSYLGLYCERATE ON THE THERMAL UNFOLDING OF RECOMBINANT NUCLEASE A FROM STAPHYLOCOCCUS AUREUS STUDIED BY PICOSECOND TIME-RESOLVED FLUORESCENCE AND CALORIMETRY

2-O-alpha-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper)thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, pico-second time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A (SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 m potassium mannosylglycerate caused an increase of 7 degrees C in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65 degrees C and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed.

Effect of specific growth rate on the production of a recombinant nuclease by Escherichia coli

The effect of specific growth rate μ on the production of Vibrio vulnificus nuclease was investigated in fed-batch cultures of a high-yield recombinant Escherichia coli. A pseudo-exponential feeding method for controlling μ was employed to obtain the desired data. It was found that the nuclease content of cells γ P varied with μ, with a maximum occurred at μ=0.05 h −1; however, the specific nuclease production rate q p increased with increasing μ. The synthesis of nuclease was dependent on μ, irrespective of the feeding methods. When concerning the compromise between the nuclease yield and its production rate, the linear-gradient feeding method, being simple and adaptable, was shown to be adequate for the nuclease production.

Involvement of conserved histidine, lysine and tyrosine residues in the mechanism of DNA cleavage by the caspase-3 activated DNase CAD.

The caspase-activated DNase (CAD) is involved in DNA degradation during apoptosis. Chemical modification of murine CAD with the lysine-specific reagent 2,4,6-trinitrobenzenesulphonic acid and the tyrosine-specific reagent N-acetylimidazole leads to inactivation of the nuclease, indicating that lysine and tyrosine residues are important for DNA cleavage by this enzyme. The presence of DNA or the inhibitor ICAD-L protects the enzyme from modification. Amino acid substitution in murine CAD of lysines and tyrosines conserved in CADs from five different species leads to variants with little if any catalytic activity, but unaltered DNA binding (K155Q, K301Q, K310Q, Y247F), with the exception of Y170F, which retains wild-type activity. Similarly, as observed for the previously characterised H242N, H263N, H308N and H313N variants, the newly introduced His-->Asp/Glu or Arg exchanges lead to variants with <1% of wild-type activity, with two exceptions: H313R shows wild-type activity, and H308D at pH 5.0 exhibits approximately 5% of wild-type activity at this pH. Y170F and H313R produce a specific pattern of fragments, different from wild-type CAD, which degrades DNA non-specifically. The recombinant nuclease variants produced in Escherichia coli were tested for their ability to form nucleolytically active oligomers. They did not show any significant deviation from the wild-type enzyme. Based on these and published data possible roles of the amino acid residues under investigation are discussed.

A Role of the Ca2+/Mg2+-dependent Endonuclease in Apoptosis and Its Inhibition by Poly(ADP-ribose) Polymerase

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca(2+)- and Mg(2+)-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.

Characterization of Serratia marcescens nuclease isoforms by plasma desorption mass spectrometry

Isoforms of Serratia marcescens nuclease found in the natural nuclease produced by S. marcescens and in recombinant nuclease produced by Escherichia coli were structurally characterized by peptide mapping using plasma desorption mass spectrometry. The nuclease isoforms produced and secreted from S. marcescens B10M1, which are present in much greater amounts than in S. marcescens W225 nuclease produced by E. coli, were characterized completely and the information used to facilitate characterization of the recombinant nuclease isoforms. After purification of the nuclease the isoforms were separated on a DEAE-cellulose anion-exchange column and then digested with endoproteinase Lys-C. The peptides generated were isolated by reverse-phase HPLC and their molecular masses determined by plasma desorption mass spectrometry. Comparison of the peptides from the native nuclease, Sm2, and the two isoforms, Sm1 and Sm3, revealed that they differed only in the N-terminus, the latter being found to lack three amino acids in Sm1 and one amino acid in Sm3. No interior post-translational changes were found in either of the three isoforms. Using this information we were able to confirm that Sm1, the isoform lacking three amino acids, was also present in very small amounts in recombinant S. marcescens W225 nuclease produced and excreted by E. coli.

High-level expression of staphylococcal nuclease R gene in Escherichia coli

Staphylococcal nuclease R, an analogue of nuclease A, was overproduced under the transcriptional control of the bacteriophage lambda P RP L promoters regulated by temperature sensitive repressors. The expression level reached 200–300 mg l −1 and showed little host dependence in different strains. The investigations of the recombinant nuclease R have revealed that the amino terminal formyl methionine residue of the nuclease is precisely processed, the protein consists of 155 amino acid residues. The experiment shows that the pBV221-DH5α is a quite suitable vector-host system for high-level expression and precise processing of heterologous genes in Escherichia coli. The comparative studies between the codons used in the staphylococcal nuclease R gene and the optimal codon usage in E. coli indicate that high level expression of heterologous genes in E. coli may not always require a high degree of codon usage bias.