PCR-based gene synthesis to produce recombinant proteins for crystallization

<p>Abstract</p> <p>Background</p> <p>Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves.</p> <p>Results</p> <p>A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille) domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA). The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to <it>in vivo </it>homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in <it>E. coli </it>and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis.</p> <p>Conclusion</p> <p>We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.</p

Thermococcus Thioreducens sp. Nov., a Novel Hyperthermophilic, Obligately Sulfur-reducing Archaeon from a Deep-sea Hydrothermal Vent

A hyperthermophilic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P was isolated from black smoker chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2 N, 33.9 W). The cells of strain OGL-20P(sup T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within the pH range 5.0-8.5 (optimum pH 7.0), NaCl concentration range 1-5 % (w/v) (optimum 3%), and temperature range 55-94 C (optimum 83-85 C). The novel isolate is strictly anaerobic and obligately dependent upon elemental sulfur as an electron acceptor, but it does not reduce sulfate, sulfite, thiosulfate, iron (III) or nitrate. Proteolysis products (peptone, bacto-tryptone, casamino-acids, and yeast extract) are utilized as substrates during sulfur-reduction. Strain OGL-20P(sup T) is resistant to ampicillin, chloramphenicol, kanamycin, and gentamycin, but sensitive to tetracycline and rifampicin. The G+C content of DNA is 52.9 mol%. The 16S rRNA gene sequence analysis revealed that strain OGL-20P(sup T) is closely related to Thermococcus coalescens and related species, but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, we conclude that strain OGL-20P(sup T) represents a new separate species within the genus Thermococcus, and propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(sup T) (= ATCC BAA-394(sup T) = JCM 12859(sup T) = DSM 14981(sup T))

Common Security of the Czech Republic and Slovakia Airspace by JAS-39 GRIPEN Fighter Aircrafts

Diplomová práce se zabývá problematikou ochrany vzdušného prostoru České republiky a Slovenska nadzvukovými letouny JAS-39 Gripen. První část práce je zaměřena na teoretickou rovinu obrany jako služby v obecném zájmu nehospodářské povahy a na vybrané pojmy z oblasti obrany a bezpečnosti státu. Druhá část se zabývá komparací Armády České republiky a Ozbrojených sil Slovenské republiky se zaměřením na vzdušné síly a možnosti ochrany vzdušného prostoru pomoci nadzvukových bojových letounů obou zemí. Třetí částí je zhodnocení možnosti pořízení a provozování nadzvukových letounů JAS-39 Gripen Vzdušnými silami Ozbrojených sil Slovenské republiky, které vychází z předchozího zkoumání a zároveň přibližuje možnosti zabezpečení společného vzdušného prostoru obou zemí. V poslední části jsou doporučení pro společné zabezpečení vzdušného prostoru obou zemí, vycházející z jednotlivých možností, ale i se stávající bezpečnostní a ekonomické situace.This thesis deals with the airspace protection of Czech Republic and Slovakia by JAS-39 Gripen supersonic aircrafts. The first part is focused on the theoretical level of defense as a service of general non-economic interest and certain terms of defense and national security. The second part deals with the comparsion of the Czech Army and Slovak Army with focuse on options of airspace protection by supersonic fighter aircrafts of both countries. The third part evaluates the possibility of acquisition and usage JAS-39 supersonic aircraft by the Slovak Air Force which is based on previous research and shows how to secure the common airspace of both countries. In the final chapter are recommendations for common airspace security of both countries based on each option, but also on current security and economic situation.153 - Katedra veřejné ekonomikyvelmi dobř

Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies

High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing

Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency

PCR-based gene synthesis to produce recombinant proteins for crystallization-1

Ted initially to sequence analysis of the synthesized product to detect nucleotide errors. Error correction can be conducted in two ways using oligonucleotide primers (30–35 nucleotides long) that are designed to include the correcting nucleotide (when needed) at the midpoint. First, mutagenic primers are targeted exclusively against the assembled synthetic gene (route A). Two, three and four pairs of primers are required to correct one, two and three point mutation (ptm) sites respectively. DNA fragments are amplified by two or all primer pairs F1-R1, F2-R2, F3-R3 and F1-R4 in separate reactions. The terminal primers have overlapping homologous regions with that of the targeted plasmid vector. Reaction products are mixed for transformation into an appropriate cell host. The second approach involves the amplification of the plasmid vector (routes B-D). To remove 3 point mutations, two correcting primers, reverse-complement of each other, are designed at each mutation site, with the correcting nucleotide being at the midpoint of each primer. DNA fragments are amplified by PCR using primer pairs F1-R1, F2-R2 and F3-R3 respectively in 3 separate reactions (D). Two pairs of primers are similarly used for 2 point mutations involving only 2 separate reactions (C). Single site error correction requires a non-mutagenic primer pair corresponding to a sequence in the vector backbone in addition to the correcting primer set such that 2 fragments are generated (as if 2 corrections were being made). Products of the correcting reactions are retransformed into competent cells for plasmid isolation and sequencing. Upon verification of error free clones, the plasmids are then transformed into an appropriate host cell for protein expression.<p><b>Copyright information:</b></p><p>Taken from "PCR-based gene synthesis to produce recombinant proteins for crystallization"</p><p>http://www.biomedcentral.com/1472-6750/8/44</p><p>BMC Biotechnology 2008;8():44-44.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2408586.</p><p></p

PCR-based gene synthesis to produce recombinant proteins for crystallization-0

Es 2) against standard molecular markers (lanes 1) containing myosin, phosphorylase, BSA, glutamic dehydrogenase, alcohol dehydrogenase, carbonic anhydrase, myoglobin red, lysozyme, aprotinin and insulin B chain on a 11% polyacrylamide gel. Approximately 15 μg of purified protein was loaded in each gel (lanes 2) and visualized with Coomassie brilliant blue. The arrows indicate recombinant proteins at about 15 kDa and 70 kDa for PAZ and PolA respectively. The proteins were estimated to be more than 90% homogeneous. Crystals of PAZ and PolA are shown in the bottom panels along with their corresponding space group and unit cell parameters as determined by preliminary X-ray analysis.<p><b>Copyright information:</b></p><p>Taken from "PCR-based gene synthesis to produce recombinant proteins for crystallization"</p><p>http://www.biomedcentral.com/1472-6750/8/44</p><p>BMC Biotechnology 2008;8():44-44.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2408586.</p><p></p