Construction of bionanoparticles with the use of a recombinant DNA vector-enzymatic system, containing artificial poliepitopic proteins, for the delivery of new generation vaccines

DNA/RNA amplification technologies, such as the Polymerase Chain Reaction have revolutionized modern biology, medical diagnostics and forensic analyses, among others. A number of alternative nucleic acids amplification methods have been developed, tailored to specific applications. Here we present a refined version of a DNA fragment amplification technology, which enables the construction of ordered concatemers in a head-to-tail-orientation. A very high number of DNA segments, at least 500 copies, can be consecutively linked. Other key features include: (i) the application of a dedicated vector-enzymatic system, including selected subtype IIS restriction endonucleases, which has been designed to automatically generate long Open Reading Frames and (ii) an amplification-expression vector with a built-in strong transcription promoter along with optimal translation initiation signals, which allow for a high level of expression of the constructed artificial poliepitopic protein. This highly advanced technology makes it possible to obtain ordered polymers of monomeric, synthetic or natural, DNA far beyond the capabilities of current chemical synthesis methods. The constructed poliepitopic proteins are further used for construction of several types of nanoparticles, including inclusion bodies and bacteriophages, containing multiple genetic fusion with poliepitopic proteins.The technology offers significant advances in a number of scientific, industrial and medical applications, including new vaccines and tissue pro-regenerative methods. The technology is protected by an international patent application and is available for licensing. Acknowledgments: project was supported by National Center for Research and Development, Warsaw, Poland, grant no STRATEGMED1/235077/9/NCBR/2014 and POIG.01.04.00-22-140/12; Jagiellonian Center for Innovation, Krakow, Poland; SATUS VC, Warsaw, Poland and BioVentures Institute Ltd, Poznan, Poland

Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb <i>Geobacillus stearothermophilus</i> bacteriophage TP-84 and its classification in the new <i>Tp84virus</i> genus

<div><p>Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the <i>Siphoviridae</i>, and infects a number of thermophilic <i>Geobacillus</i> (<i>Bacillus</i>) <i>stearothermophilus</i> strains. Its’ 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host’s transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the <i>Siphoviridae</i> family. Thus a taxonomic proposal of a new <i>Tp84virus</i> genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in <i>Escherichia coli</i>, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.</p></div

Electron microscopy image of the purified TP-84 bacteriophage.

<p>Purified TP-84 sample loaded onto 300 mesh copper grid (Sigma), covered with 2% collodion (Sigma), sprayed with carbon and stained with 2% uranyl acetate (BDH Chemicals). Visualised with a Tecnai G2 Spirit BioTWIN TEM set at 120 kV. Pictures were captured with a Veleta CCD camera.</p

Genome organization of the thermophilic, 47.7-kb bacteriophage TP-84.

<p>Putative genes, encoding proteins with assigned biological function, are marked with red arrows. Genes with assigned function confirmed by proteomic analysis are marked with orange arrows. Genes without assigned biological function are marked with black arrows. P—putative host-dependent promoter, T–Rho-independent terminator, SSB–single-stranded DNA-binding protein. The scheme was created using SnapGene software (<a href="http://www.snapgene.com/" target="_blank">http://www.snapgene.com</a>) and further modified.</p

Experimental validation of TP-84 proteins biosynthesized during the infection time-course.

<p>Samples for MS analysis were taken from <i>G</i>. <i>stearothermophilus</i> cultures at time intervals: U, uninfected control prior to infection; 0, sample taken immediately upon TP-84 addition; 5, 5 min after infection; 10, 10 min; 15, 15 min; 20, 20 min; 25, 25 min; 30, 30 min. Panel A-D show graphs for each TP-84 protein detected in the culture samples, grouped according to their function. Panel A) DNA replication, recombination, transcription-related and nucleotide metabolism protein. Panel B). Structural proteins and packaging. Panel C) Cell wall and membrane degrading proteins. Panel D) Unknown function proteins.</p

Experimental validation of TP-84 SSB and structural proteins.

<p>Panels A-C. Expression, purification and functional assay of TP-84 SSB-His₆ protein. Lanes M1, molecular weight protein marker, LMW-SDS Marker (GE Healthcare). Panel A. SDS-PAGE analysis of the recombinant <i>E</i>. <i>coli</i> TOP10 [pBADMycHisA-TP-84_SSB] cells induction time course. Lane 1, <i>E</i>. <i>coli</i> TOP10 [pBADMycHisA-TP-84_SSB] cells prior to arabinose induction; lane 2, 2 h after induction; lane 3, 4 h after induction; lane 4, 16 h after induction. Panel B. Metal-affinity purification of TP-84 SSB-His₆ protein. Lane 1, purified TP-84 SSB-His₆ protein. Panel C. PCR assay of DNA-binding capabilities of TP-84 SSB-His₆ protein. Lane M2, molecular weight DNA marker, 100-bp Plus (Thermo Scientific, USA); lane 1, PCR reaction without addition of TP-84 SSB-His₆ protein; lane 2, 0.36 μg of TP-84 SSB-His₆ protein added; lane 3, 0.72 μg; lane 4, 1.08 μg; lane 5, 1.44 μg; lane 6, 2.16 μg; lane 7, 2.88 μg; lane 8, 3.6 μg. Panel D. SDS-PAGE analysis of the proteins of purified TP-84 bacteriophage. Protein bands yielding MS results of high credibility are assigned to the matching TP-84 CDSs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195449#pone.0195449.s005" target="_blank">S5 File</a>).</p

Experimental validation of TP-84 proteins biosynthesized during the infection time-course.

<p>Samples for MS analysis were taken from <i>G</i>. <i>stearothermophilus</i> cultures at time intervals: U, uninfected control prior to infection; 0, sample taken immediately upon TP-84 addition; 5, 5 min after infection; 10, 10 min; 15, 15 min; 20, 20 min; 25, 25 min; 30, 30 min. Panel A-D show graphs for each TP-84 protein detected in the culture samples, grouped according to their function. Panel A) DNA replication, recombination, transcription-related and nucleotide metabolism protein. Panel B). Structural proteins and packaging. Panel C) Cell wall and membrane degrading proteins. Panel D) Unknown function proteins.</p

Phylogenetic trees for TP-84 bacteriophage.

<p>A) Phylogenetic tree constructed using the large subunit terminase (TerL). B) Phylogenetic tree constructed using thymidylate synthase (Ts) proteins. The trees do not include all <i>Geobacillus</i> phage proteins since the differences between all the sequences renders the trees unreliable.</p

The GC-skew distribution over the genome of TP-84.

<p>The bacteriophage genome was analysed using the default settings of GC Content Calculator (Biologics International Corp, Indianapolis, USA; <a href="http://www.biologicscorp.com/tools/GCContent/" target="_blank">http://www.biologicscorp.com/tools/GCContent/</a>; blue).</p