Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.     

An EBV‐based plasmid can replicate and maintain in stem cells

Viral vectors have a wide range of applications in biology, particularly in gene therapy. Based on their integration capacity, viral vectors are classified as either integrating or non‐integrating vectors. Although integrating vectors, such as lentivectors, have the ability to direct prolonged expression of exogenous genes, manipulation of the host genome is an inappropriate feature of these gene delivery tools. Non‐integrating vectors, such as episomal replicating plasmids, can replicate and persist in host cells for long periods without any chromosomal interruption. These advantages made them good tools for gene induction purposes in gene therapy and basic studies. Due to the necessity of gene induction in stem cells for study of mammalian development and targeted differentiation, the use of integrating vectors for prolonged expression of genes of interest has been developed. Application of replicating plasmids can overcome some drawbacks associated with integrating vectors, although replication and maintenance of these plasmids can differ between cell types. Previously, it has been shown that such plasmids can be maintained in human embryonic stem cells for more than one month, but the rate of the plasmid replication during the host cell cycle has not been elucidated. In the present study, we showed that an EBV‐based plasmid can replicate simultaneously with host in pluripotent and multipotent human and mouse stem cells and can be sustained for long time periods in dividing cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1579–1585, 2015     

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.     

Phenomenology and molecular mechanisms of Shigella sonnei dissociation: its variability in model in vivo and in vitro systems

The dynamics of the accumulation of phase II S. sonnei in the infectious process in germ-free rats, during cultivation and in subcultures was studied. The in vivo variability of S. sonnei showed a sharply defined phasic character of the process with the periods of the apparent absence of phase II, the increase of its occurrence and stabilization with the preservation of S-forms in minor amounts. Kanamycin-resistant phase II accumulated in vivo more rapidly than in vitro. Cultivation with sodium desoxycholate was found to accelerate the process of disassociation. The characteristic feature of all dissociants detected in all systems was their plasmid profile: in phase I, plasmids of 120 and 60 Md, as well as small plasmids, were detected; in phase II disassociants, plasmid with a molecular weight of 120 Md was absent. The restriction analysis of total plasmid DNA by means of restrictase EcoR1 showed the absence of 3 fragments in phase II while other 13 fragments were retained. These results open prospects for the molecular cloning of the gene antigen of phase II.     

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.     

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Construction of shuttle vectors for cloning inEscherichia coli andAcetobacter pasteurianus

New cloning vectors were prepared with the aid of a large plasmid isolated fromAcetobacter pasteurianus and from plasmids pBR322 and pUC4-KAPA. Of the prepared cloning vectors, pACK5 contains a gene coding for kanamycin resistance, pACT7 and pACT71 contain a gene coding for tetracycline resistance and vector pACG3 with a gene coding for both kanamycin and tetracycline resistance. The vectors prepared only contained the beginning of replication from the pAC1 plasmid and possessed the ability to replicate withinE. coli andA. pasteurianus. The vectors are highly stable in both strains and during the 5-d cultivation under nonselective conditions are not eliminated.     

Defining components of the chromosomal origin of replication of the hyperthermophilic archaeon Pyrococcus furiosus needed for construction of a stable replicating shuttle vector

We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus, plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Formation of plasmid pBR322 oligomers as depending on a tetracycline concentration

Escherichia coli K12 strains containing the plasmid pBR322 often show varying contents of plasmid oligomers, in which the monomer units are arranged in tandem. When the concentration of the plasmid-selective antibiotic tetracycline in the medium becomes increased selection of cells containing largely higher oligomers occurs. The number of monomer units organized in the oligomers increases with tetracycline concentration. recA- mutants are unable to generate oligomers under the same conditions and show lower tetracycline resistance. This observations suggest a selective advantage of oligomer containing cells in the presence of tetracycline as a result of higher gene dosage. But E. coli cells transformed with monomers, dimers, trimers, as well as tetramers of pBR322 are characterized by roughly the same plasmid DNA content as well as plasmid coded beta-lactamase and resistance to tetracycline.     

Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis

We report here the first use of directed mutagenesis in Methanosarcina acetivorans C2A. The method employs homologous recombination-mediated gene replacement and was used to construct a variety of proline auxotrophs with mutations in the proABC locus. Each mutation was also complemented in trans with autonomously replicating Methanosarcina-Escherichia plasmid shuttle vectors.     

Constructing recombinant herpesvirus BAC vectors with mating-assisted genetically integrated clone method

The large capacity of pseudorabies virus (PRV) for foreign DNA and broad host range make it a prospective tool for the preparation of vaccines and agents of gene and tumour therapy. Here we introduced a cloning strategy that facilitates construction of recombinant PRV?CBAC vectors based on mating-assisted genetically integrated clone (MAGIC). The target gene was cloned into a small conditionally replicating donor plasmid, followed by shuffling to a recipient PRV?CBAC plasmid in vivo of Escherichia coli through MAGIC. The average efficiency of successful clones was 89%. Moreover, permanent integration of unwanted sequences was avoided.     

Cooperative binding mode of the inhibitors of R6K replication, pi dimers

The replication initiator protein, π, plays an essential role in the initiation of plasmid R6K replication. Both monomers and dimers of π bind to iterons in the γ origin of plasmid R6K, yet monomers facilitate open complex formation, while dimers, the predominant form in the cell, do not. Consequently, π monomers activate replication, while π dimers inhibit replication. Recently, it was shown that the monomeric form of π binds multiple tandem iterons in a strongly cooperative fashion, which might explain how monomers outcompete dimers for replication initiation when plasmid copy number and π supply are low. Here, we examine cooperative binding of π dimers and explore the role that these interactions may have in the inactivation of γ origin. To examine π dimer/iteron interactions in the absence of competing π monomer/iteron interactions using wild-type π, constructs were made with key base changes to each iteron that eliminate π monomer binding yet have no impact on π dimer binding. Our results indicate that, in the absence of π monomers, π dimers bind with greater cooperativity to alternate iterons than to adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by π dimers.     

Gene replacement in mycobacteria by using incompatible plasmids

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.     

Production of viral vectors using recombinase-mediated cassette exchange

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.     

AT-rich sequences from the arbuscular mycorrhizal fungus Gigaspora rosea exhibit ARS function in the yeast Saccharomyces cerevisiae

Autonomous replicating sequences are DNA elements that trigger DNA replication and are widely used in the development of episomal transformation vectors for fungi. In this paper, a genomic library from the mycorrhizal fungus Gigaspora rosea was constructed in the integrative plasmid YIp5 and screened in the budding yeast Saccharomyces cerevisiae for sequences that act as ARS and trigger plasmid replication. Two genetic elements (GrARS2, GrARS6) promoted high-rates of yeast transformation. Sequence analysis of these elements shows them to be AT-rich (72-80%) and to contain multiple near-matches to the yeast autonomous consensus sequences ACS and EACS. GrARS2 contained a putative miniature inverted-repeat transposable element (MITE) delimited by 28-bp terminal inverted repeats (TIRs). Disruption of this element and removal of one TIR increased plasmid stability several fold. The potential for palindromes to affect DNA replication is discussed.     

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.