Cloning and expression of the Propionibacterium shermanii methylmalonyl-CoA epimerase gene in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Genomic DNA was isolated from Propionibacterium shermanii (52W). A 454 bp DNA fragment coding for the methylmalonyl-CoA epimerase (EC 5.1.99.1, subsequently referred to as epimerase) was amplified from genomic DNA by the polymerase chain reaction using primers designed from the known DNA sequence of the gene. The P. shermanii epimerase gene was ligated into the 2.47 kbp expression vector pT7-7. The ligation reaction mixture was transformed into electroporation competent E.coli XL1-Blue cells. Plasmid DNA prepared from several transformants was analysed, by agarose gel electrophoresis of restriction enzyme digestions, and transformed into E.coli SRP84/pGP1-2 cells to identify potential epimerase expression constructs (pTEEX) by heat shock induction. The insert DNA of one of the putative pTEEX epimerase constructs was fully sequenced and shown to be identical to the known DNA sequence of the epimerase gene described by Davis (1987). Using the sequenced expression construct pTEEX, recombinant epimerase was expressed to 20-35% of the total cell protein in the protease deficient E.coli strain SRP84 using the dual plasmid expression system of Tabor and Richardson (1985). The recombinant epimerase was ~95-100% soluble in E.coli. The recombinant epimerase and the 'wild-type' epimerase produced by P. shermanii were purified using the procedures developed for the 'wild-type' epimerase. The addition of a heat-treatment step (70°C for 15 min) early in the purification of the recombinant enzyme successfully exploited the unusually high thermostability of the epimerase protein. The epimerase protein was found to have an anomalously low electrophoretic mobility in a modified Laemmli discontinuous Tris-glycine alkaline buffer system for SDS-PAGE gels compared to the Weber and Osborn continuous phosphate buffer system. Using the latter system, a subunit molecular weight of 16.6 kDa was obtained. This is consistent with the molecular weight of 16.72 kDa (methionine on) calculated from the inferred amino acid sequence. The N-terminal sequence of the purified 'wild-type' and recombinant epimerases were identical although only half of N-terminal methionine residues were removed from the recombinant protein. The subunit molecular weight, specific activity, activation by divalent metal ions and behaviour in crystallization trials of the 'wild-type' and recombinant epimerases were very similar. Recombinant epimerase crystals were grown in a buffer containing 0.2 M ammonium acetate and 0.1 M citrate, pH 5.6, containing 30% PEG 4000 as precipitant. These crystals were relatively poorly ordered and diffracted to only 4.5 Ǻ resolution, but crystals of the recombinant epimerase that diffract to 2.6Ǻ can be grown under appropriate conditions

Coherent band pathways between knots and links

We categorise coherent band (aka nullification) pathways between knots and 2-component links. Additionally, we characterise the minimal coherent band pathways (with intermediates) between any two knots or 2-component links with small crossing number. We demonstrate these band surgeries for knots and links with small crossing number. We apply these results to place lower bounds on the minimum number of recombinant events separating DNA configurations, restrict the recombination pathways and determine chirality and/or orientation of the resulting recombinant DNA molecules

Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5′-Olig2cDNA-IRES-dsRed2-3′, we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells

Recombinant subunit vaccines against Neospora caninum

The intracellular protozoan parasite Neospora caninum is an important cause of bovine abortion and congenital infection in many countries. Currently there is no effective control method available, and development of a vaccine has been suggested as a possible strategy to prevent the disease neosporosis. In this thesis an experimental recombinant subunit vaccine, consisting of the N. caninum antigen NcSRS2 and immunostimulating complexes (iscoms), was evaluated. The iscom is an adjuvant formulation into which antigens can be incorporated by hydrophobic interactions. Many recombinant proteins are hydrophilic, and special methods are needed for binding them to the iscoms and to achieve optimal adjuvant effects. A novel method to combine recombinant proteins with iscoms utilizing the strong interaction between biotin and streptavidin was evaluated. Recombinant NcSRS2 was expressed in Escherichia coli as two different fusion proteins. One of them was biotinylated in E. coli and was bound to streptavidin-coated iscom matrix (iscom particles without any antigen). The other fusion protein contained recombinant streptavidin and was bound to biotinylated iscom matrix. These two strategies both resulted in efficient binding to the iscom matrix. Thus, this method might offer a convenient and effective alternative for association of other hydrophilic recombinant antigens with iscoms for evaluation in immunisation experiments. Both the immunogenicity and the protective effect of the NcSRS2-iscoms were investigated by immunisation and challenge infection of mice. A real-time PCR assay was developed, and used for quantification of parasite DNA in the brain and blood of infected mice. The NcSRS2-iscoms induced production of antibodies that recognised NcSRS2 of parasite origin, suggesting that the conformation of the recombinant protein was similar to that of the native antigen. Antigen-specific cellular responses were also induced as demonstrated by in vitro proliferation and cytokine production. After challenge infection, the immunised mice had significantly lower levels of N. caninum DNA in their brains and blood than did non-immunised control mice. These mice were also less affected by the disease, as judged by clinical symptoms and changes in body weight. These results, together with results from other studies, indicate that recombinant NcSRS2 might be considered as a potential candidate antigen for a future subunit vaccine against N. caninum infection in cattle

Screening for the optimal siRNA targeting a novel gene (HA117) and construction and evaluation of a derivative recombinant adenovirus

We found a novel gene named as HA117 in our previous research. At this study, we screened for an optimal siRNA targeting the novel gene HA117 using the pSOS-HUS method, verified the results of pSOS-HUS siRNA screening for optimal affinity for the target gene, and constructed and evaluated a recombinant adenovirus carrying the DNA template for transcription of the optimal HA117 siRNA. The pSOS-HUS vector method was successfully utilized as a rapid and effective screen for an optimal siRNA for a target gene. Among five pairs of DNA templates, siRNA transcribed from HAi5 gave the strongest interference with the novel gene HA117; a HAi5-carrying recombinant adenovirus (Ad-HAi5) was successfully constructed and evaluated, laying a foundation for the further study of HA117 gene function with RNAi technology

Ciz1 cooperates with cyclin-A-CDK2 to activate mammalian DNA replication in vitro

Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A-CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order

Recombinant DNA Molecules of Bacteriophage phi X174

phi X174 DNA structures containing two different parental genomes were detected genetically and examined by electron microscopy. These structures consisted of two monomeric double-stranded DNA molecules linked in a figure 8 configuration. Such DNA structures were observed to be formed preferentially in host recA+ cells or recA+ cell-free systems. Since the host recA+ allele is required for most phi X174 recombinant formation, we conclude that the observed figure 8 molecules are intermediates in, or end products of, a phi X174 recombination event. We propose that recombinant figure 8 DNA molecules arise as a result of "single-strand aggression," are stabilized by double-strand "branch migration," and represent a specific example of a common intermediate in genetic recombination