Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases.

BACKGROUND: The discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called 'Point-of-Care tests'. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited. RESULTS: We have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8-9 and 25-110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25-37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme. Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes. CONCLUSIONS: Substituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.

Characterization of an intertidal zone metagenome oligoribonuclease and the role of the intermolecular disulfide bond for homodimer formation and nuclease activity.

The gene encoding MG Orn has been identified from a metagenomic library created from the intertidal zone in Svalbard and encodes a protein of 184 amino acid residues. The mg orn gene has been cloned, recombinantly expressed in Escherichia coli, and purified to homogeneity. Biochemical characterization of the enzyme showed that it efficiently degrades short RNA oligonucleotide substrates of 2mer to 10mer of length and has an absolute requirement for divalent cations for optimal activity. The enzyme is more heat-labile than its counterpart from E. coli and exists as a homodimer in solution. The crystal structure of the enzyme has been determined to a resolution of 3.15 Å, indicating an important role of a disulfide bridge for the homodimer formation and as such for the function of MG Orn. Substitution of the Cys110 residue with either Gly or Ala hampered the dimer formation and severely affected the enzyme's ability to act on RNA. A conserved loop containing His128-Tyr129-Arg130 in the neighboring monomer is probably involved in efficient binding and processing of longer RNA substrates than diribonucleotides.

Characterization of an antiserum against Atlantic cod (Gadus morhua L.) g-type lysozyme.

In this study we describe the production and characterization of an antiserum against recombinant g-type lysozyme derived from Atlantic cod. This is also the first initial analyses of g-type lysozyme protein expression in tissues of Atlantic cod. Recombinant expression and purification of cod g-type lysozyme was used for immunization to rabbit and the rabbit sera were analysed for anti g-type lysozyme antibodies using enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry. ELISA results showed that antibody titres were mounted between 12,800 and 25,600 as measured at an optical density corresponding to 50% of the maximal level. By Western blot analysis the rabbit immune serum detected a single ∼23 kDa band representing the size of the injected antigen, in both spleen and head kidney homogenates from the Atlantic cod. Immunohistochemisrty detected the native folded g-type lysozyme in tissues and revealed that g-type lysozyme positive cells were observed in haematopoietic tissue of the head kidney and in red pulp of spleen. In conclusion, the rabbit anti g-type lysozyme immune sera was developed and is effectively utilized for ELISA, Western analysis as well as for immunohistochemistry. This has allowed us to obtain new knowledge about this protein regarding localization and distribution in cod tissue.

Interleukin-17D in Atlantic salmon (Salmo salar): molecular characterization, 3D modelling and promoter analysis.

IL-17 is a proinflammatory cytokine that plays an important role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. Much work on IL-17 has been done in humans and higher vertebrates while little work has been conducted in lower vertebrates including fish. In this study, we have cloned and characterized the full-length cDNA and genomic sequence of IL-17D from Atlantic salmon. The Atlantic salmon IL-17D (AsIL-17D) cDNA possessed an open reading frame of 621 bp encoding a putative protein of 206 aa with a predicted molecular weight of 23 kDa. The AsIL-17D gene has two exons and one intron showing the same (genome) organisation compared to zebrafish IL-17D. The encoded protein showed 97.6-48.8% identities to other IL-17D homologues, eight conserved cysteine residues were found within this group. Conserved residues believed to be important in receptor binding were also confirmed in salmon IL-17D by homology modelling. Phylogenetic analysis also confirmed the close relationship with other IL-17D homologues. Functional characterization of the 5' flanking region indicated that the region between -1552 and -150 contained sufficient elements for promoter activity. Tissue expression studies by real-time PCR showed a predominant expression of IL-17D transcript in gonads, skin, intestine, thymus of Atlantic salmon. The involvement of IL-17D during proinflammatory responses was demonstrated by investigating the time-dependent expression profile of IL-17D in head kidney and spleen following intraperitoneal injection of live Aeromonas salmonicida, LPS, and beta-glucan. This study provides further evidence for the existence of distinct homologue of IL-17D isoform in fish showing early expression induced by immunostimulants and bacterial infection that supports the fact that IL-17D is regulated by inflammatory processes in fish.

Characterisation and expression analysis of the interleukin genes, IL-1beta, IL-8 and IL-10, in Atlantic cod (Gadus morhua L.).

The mammalian interleukins IL-1beta and IL-8 are important pro-inflammatory cytokines often used as markers of an activated inflammatory response, while IL-10 is regarded as an anti-inflammatory cytokine and plays a crucial role in the regulation of inflammation. Few cytokines from gadoid fish have been described, and herein the sequence and expression of these interleukin genes are studied in Atlantic cod (Gadus morhua L.). IL-1beta, IL-8 and IL-10 from cod show similarities in gene organisation and predicted protein sequences, compared to counterpart genes in other teleosts. Gene expression was studied using quantitative real time PCR in response to several treatments both in vitro and in vivo. In adherent head kidney cells, infectious pancreatic necrosis virus (IPNV) and lipopolysaccharide (LPS) significantly stimulated gene expression of IL-1beta. The expression of IL-1beta was not increased after treatment with a viral imitator (poly I:C), and neither IL-8 nor IL-10 responded to any of these agents in vitro. However, in vivo administrated poly I:C and formalin-killed Vibrio anguillarum (In-V.ang) induced interleukin expression, varying in intensity between different organs. IL-1beta and IL-10 gene expression profiles showed an opposite induction pattern in the in vivo experiments. Injection of In-V.ang highly induced IL-1beta expression, while a low induction was evident for IL-10, whereas the opposite was observed after injection of poly I:C. This pattern was particularly marked in spleen, where also IL-8 followed the expression pattern of IL-1beta. The opposite expression profiles indicate a suppressive role for IL-10 on the transcription of IL-1beta, and to a lesser extent on IL-8 transcription. Along with the identification of important promoter regulatory motives, these results provide tools for studying inflammatory responses in cod.

Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp.

The gene encoding a peptidase that belongs to the proteinase K family of serine peptidases has been identified from a psychrotrophic Serratia sp., and cloned and expressed in Escherichia coli. The gene has 1890 base pairs and encodes a precursor protein of 629 amino acids with a theoretical molecular mass of 65.5 kDa. Sequence analysis suggests that the peptidase consists of a prepro region, a catalytic domain and two C-terminal domains. The enzyme is recombinantly expressed as an active approximately 56 kDa peptidase and includes both C-terminal domains. Purified enzyme is converted to the approximately 34 kDa form by autolytic cleavage when incubated at 50 degrees C for 30 min, but retains full activity. In the present work, the Serratia peptidase (SPRK) is compared with the family representative proteinase K (PRK) from Tritirachium album Limber. Both enzymes show a relatively high thermal stability and a broad pH stability profile. SPRK possess superior stability towards SDS at 50 degrees C compared to PRK. On the other hand, SPRK is considerably more labile to removal of calcium ions. The activity profiles against temperature and pH differ for the two enzymes. SPRK shows both a broader pH optimum as well as a higher temperature optimum than PRK. Analysis of the catalytic properties of SPRK and PRK using the synthetic peptide succinyl-Ala-Ala-Pro-Phe-pNA as substrate showed that SPRK possesses a 3.5-4.5-fold higher kcat at the temperature range 12-37 degrees C, but a fivefold higher Km results in a slightly lower catalytic efficiency (kcat/Km) of SPRK compared to PRK.

The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species.

Proteins from organisms living in extreme conditions are of particular interest because of their potential for being templates for redesign of enzymes both in biotechnological and other industries. The crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species has been solved to 1.8 A. The structure has been compared with the structures of proteinase K from Tritirachium album Limber and Vibrio sp. PA44 in order to reveal structural explanations for differences in biophysical properties. The Serratia peptidase shares around 40 and 64% identity with the Tritirachium and Vibrio peptidases, respectively. The fold of the three enzymes is essentially identical, with minor exceptions in surface loops. One calcium binding site is found in the Serratia peptidase, in contrast to the Tritirachium and Vibrio peptidases which have two and three, respectively. A disulfide bridge close to the S2 site in the Serratia and Vibrio peptidases, an extensive hydrogen bond network in a tight loop close to the substrate binding site in the Serratia peptidase and different amino acid sequences in the S4 sites are expected to cause different substrate specificity in the three enzymes. The more negative surface potential of the Serratia peptidase, along with a disulfide bridge close to the S2 binding site of a substrate, is also expected to contribute to the overall lower binding affinity observed for the Serratia peptidase. Clear electron density for a tripeptide, probably a proteolysis product, was found in the S' sites of the substrate binding cleft.