The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene

An Arthrobacter species FB24 gene (locus tag Arth_1007) was previously annotated as a putative intein-containing DnaB helicase of phage origin (Arsp-FB24 DnaB intein). However, it is not a helicase gene because the sequence similarity is limited to inteins. In fact, the flanking exteins total only 66 amino acids. Therefore, the intein should be referred to as the Arsp-FB24 Arth_1007 intein. The Arsp-FB24 Arth_1007 intein failed to splice in its native precursor and in a model precursor. We previously noted that the Arsp-FB24 Arth_1007 intein is the only putative Class 3 intein that is missing the catalytically essential Cys at position 4 of intein Motif F, which is one of the three defining signature residues of this class. Additionally, a catalytically essential His in position 10 of intein Motif B is also absent; this His is the most conserved residue amongst all inteins. Splicing activity was not rescued when these two catalytically important positions were ‘reverted’ back to their consensus residues. This study restores the unity of the Class 3 intein signature sequence in active inteins by demonstrating that the Arsp-FB24 Arth_1007 intein is an inactive pseudogene

A Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch

Conservation of intron and intein insertion sites: implications for life histories of parasitic genetic elements

<p>Abstract</p> <p>Background</p> <p>Inteins and introns are genetic elements that are removed from proteins and RNA after translation or transcription, respectively. Previous studies have suggested that these genetic elements are found in conserved parts of the host protein. To our knowledge this type of analysis has not been done for group II introns residing within a gene. Here we provide quantitative statistical support from an analyses of proteins that host inteins, group I introns, group II introns and spliceosomal introns across all three domains of life.</p> <p>Results</p> <p>To determine whether or not inteins, group I, group II, and spliceosomal introns are found preferentially in conserved regions of their respective host protein, conservation profiles were generated and intein and intron positions were mapped to the profiles. Fisher's combined probability test was used to determine the significance of the distribution of insertion sites across the conservation profile for each protein. For a subset of studied proteins, the conservation profile and insertion positions were mapped to protein structures to determine if the insertion sites correlate to regions of functional activity. All inteins and most group I introns were found to be preferentially located within conserved regions; in contrast, a bacterial intein-like protein, group II and spliceosomal introns did not show a preference for conserved sites.</p> <p>Conclusions</p> <p>These findings demonstrate that inteins and group I introns are found preferentially in conserved regions of their respective host proteins. Homing endonucleases are often located within inteins and group I introns and these may facilitate mobility to conserved regions. Insertion at these conserved positions decreases the chance of elimination, and slows deletion of the elements, since removal of the elements has to be precise as not to disrupt the function of the protein. Furthermore, functional constrains on the targeted site make it more difficult for hosts to evolve immunity to the homing endonuclease. Therefore, these elements will better survive and propagate as molecular parasites in conserved sites. In contrast, spliceosomal introns and group II introns do not show significant preference for conserved sites and appear to have adopted a different strategy to evade loss.</p

A Cost-Effective ELP-Intein Coupling System for Recombinant Protein Purification from Plant Production Platform

BACKGROUND: Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. METHODOLOGY/PRINCIPAL FINDINGS: To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2-4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. CONCLUSION/SIGNIFICANCE: This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry

In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii

Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type

Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics

A proposed new bacteriophage subfamily: “Jerseyvirinae”

© 2015, Springer-Verlag Wien. Based on morphology and comparative nucleotide and protein sequence analysis, a new subfamily of the family Siphoviridae is proposed, named “Jerseyvirinae” and consisting of three genera, “Jerseylikevirus”, “Sp3unalikevirus” and “K1glikevirus”. To date, this subfamily consists of 18 phages for which the genomes have been sequenced. Salmonella phages Jersey, vB_SenS_AG11, vB_SenS-Ent1, vB_SenS-Ent2, vB_SenS-Ent3, FSL SP-101, SETP3, SETP7, SETP13, SE2, SS3e and wksl3 form the proposed genus “Jerseylikevirus”. The proposed genus “K1glikevirus” consists of Escherichia phages K1G, K1H, K1ind1, K1ind2 and K1ind3. The proposed genus “Sp3unalikevirus” contains one member so far. Jersey-like phages appear to be widely distributed, as the above phages were isolated in the UK, Canada, the USA and South Korea between 1970 and the present day. The distinguishing features of this subfamily include a distinct siphovirus morphotype, genomes of 40.7-43.6kb (49.6-51.4mol% G+C), a syntenic genome organisation, and a high degree of nucleotide sequence identity and shared proteins. All known members of the proposed subfamily are strictly lytic