An intein-cassette integration approach used for the generation of a split TEV protease activated by conditional protein splicing

Ligand-induced conditional protein splicing (CPS) using a split intein allows the covalent reconstitution of a protein from two polypeptide fragments. The small molecule rapamycin binds to the fused FKBP and FRB dimerizer domains and thereby induces folding of the split intein, which then removes itself in the trans-splicing reaction. CPS has great potential for the experimental control of protein activity in living cells, however, only one such example was reported yet. This discrepancy is due to the challenging reconstitution of a protein from two inactive fragments because of folding, stability, and solubility issues. Moreover, in CPS the split intein must be active in the specific sequence context. We here report the novel concept, design, and application of a CPS cassette for facile target gene modification to identify active split intein insertion sites. The CPS cassette encodes the split intein and dimerizer domain gene fragments as well as a selectable genetic marker for yeast. The addition of short sequences in the PCR-amplification of the CPS cassette allowed its site-specific insertion into the target gene by homologous recombination. Our approach thus avoids the extensive DNA cloning steps typically required. By this strategy, we identified two CPS variants of the tobacco etch virus (TEV) protease that are conditionally activated by rapamycin in yeast and we show their potential for the manipulation of intracellular proteins through proteolysis events. Our results suggest that more proteins will be amenable to CPS control and that intein cassette integration is a powerful tool for the development of such conditional variants as well as for other application of cis- and trans-splicing inteins.     

GENETIC EXCHANGES OF INTEINS BETWEEN PRASINOVIRUSES (PHYCODNAVIRIDAE)

Phylogenetic diversity in the Phycodnaviridae (double‐stranded DNA viruses infecting photosynthetic eukaryotes) is most often studied using their DNA polymerase gene (PolB). This gene and its translated protein product can harbor a selfish genetic element called an “intein” that disrupts the sequence of the host gene without affecting its activity. After translation, the intein peptide sequence self‐excises precisely, producing a functional ligated host protein. In addition, inteins can encode homing endonuclease (HEN) domains that permit the possibility of lateral transfers to intein‐free alleles. However, no clear evidence for their transfer between viruses has previously been shown. The objective of this paper was to determine whether recent transfers of inteins have occurred between prasinoviruses (Phycodnaviridae) that infect the Mamiellophyceae, an abundant and widespread class of unicellular green algae, by using DNA sequence analyses and cophylogenetic methods. Our results suggest that transfer among prasinoviruses is a dynamic ongoing process and, for the first time in the Phycodnaviridae family, we showed a recombination event within an intein.     

A topA intein in Pyrococcus furiosus and its relatedness to the r-gyr intein of Methanococcus jannaschii

A new intein coding sequence was found in a topA (DNA topoisomerase I) gene by cloning and sequencing this gene from the hyperthermophilic Archaeon Pyrococcus furiosus. The predicted Pfu topA intein sequence is 373 amino acids long and located two residues away from the catalytic tyrosine of the topoisomerase. It contains putative intein sequence blocks (C, E, and H) associated with intein endonuclease activity, in addition to intein sequence blocks (A, B, F, and G) that are necessary for protein splicing. This DNA topoisomerase I intein is most related to a reverse gyrase intein from the methanogenic Archaeon Methanococcus jannaschii. These two inteins share 31% amino acid sequence identity and, more importantly, have the same insertion sites in their respective host proteins. It is suggested that these two inteins are homologous inteins present in structurally related, but functionally distinct, proteins, with implications on intein evolution and intein homing.     

SOS-inducible DNA polymerase II of E coli is homologous to replicative DNA polymerase of eukaryotes

The polB gene of Escherichia coli encodes DNA polymerase II whose role in vivo is not defined. The polB gene has been cloned and shown to be identical to a DNA damage-inducible gene dinA which is regulated by the LexA repressor. Nucleotide sequencing of polB reveals that E coli DNA polymerase II is highly homologous to replicative DNA polymerases of eukaryotes which include human DNA polymerase alpha and Saccharomyces cerevisiae DNA polymerases I, II and III. The polB gene is not required for growth, UV-repair and UV-mutagenesis.     

The Escherichia coli polB gene, which encodes DNA polymerase II, is regulated by the SOS system.

The dinA (damage inducible) gene was previously identified as one of the SOS genes with no known function; it was mapped near the leuB gene, where the polB gene encoding DNA polymerase II was also mapped. We cloned the chromosomal fragment carrying the dinA region from the ordered Escherichia coli genomic library and mapped the dinA promoter precisely on the physical map of the chromosome. The cells that harbored multicopy plasmids with the dinA region expressed very high levels of DNA polymerase activity, which was sensitive to N-ethylmaleimide, an inhibitor of DNA polymerase II. Expression of the polymerase activity encoded by the dinA locus was regulated by SOS system, and the dinA promoter was the promoter of the gene encoding the DNA polymerase. From these data we conclude that the polB gene is identical to the dinA gene and is regulated by the SOS system. The product of the polB (dinA) gene was identified as an 80-kDa protein by the maxicell method.     

Nucleotide sequence and deletion analysis of the polB gene of Escherichia coli

The polB gene encodes DNA polymerase II in Escherichia coli. The nucleotide sequence shows an open reading frame of 2,304 nucleotides coding for a protein of 88 kD. The protein initiation signal is preceded by a lexA box lying 2 nucleotides from the termination signal of araD, and begins with GUG 75 nucleotides after the termination of araD. The polB gene and the araD gene are transcribed in the same direction. Initiation of protein synthesis was confirmed by peptide sequence. We have also demonstrated that the polB sequence is lacking in some strains. We conclude that DNA polymerase II is not a required protein in the cell. Sequence comparisons show that DNA polymerase II is an alpha-like DNA polymerase.     

Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein

Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1), is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn(2+) and not Mg(2+) metal cations for activity.     

Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain

We report that the N- and C-terminal splicing domains of the intein found in the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) are capable of association in vivo and in vitro, even with key splicing residues changed to alanine (Cys(1), Asn(159), and Cys(+1) to Ala). These studies utilized the herbicide resistant form of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Salmonella typhimurium and an Escherichia coli strain with the EPSPS gene deleted from its genome (E. coli strain ER2799). EPSPS was mapped to identify potential split sites using a facile Tn7 linker scanning procedure. Forty positions were found to tolerate a five amino acid insertion while 21 sites did not, as assayed by the rescue of growth of E. coli strain ER2799. Further characterization of these sites by inserting a full length Ssp DnaE intein identified residue 235 of EPSPS as the optimal position. The EPSPS gene was then divided into amino acids 1-235 and 236-427 which were fused to residues 1-123 and 124-159 of a splicing defective Ssp DnaE intein, respectively. Expression of the EPSPS-intein fusions from separate DNA molecules conferred resistance to the herbicide glyphosate, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. As a control the split EPSPS without the intein-affinity domain did not allow cell growth. The use of an intein as an in vivo affinity domain was termed intein-mediated protein complementation (IPC). Intein fragment assembly was verified in vitro by immobilizing the C-terminal splicing domain of the Ssp DnaE intein on a resin and demonstrating that the N-terminal 235 amino acids of EPSPS only bound to the resin when fused to the N-terminal splicing domain of the Ssp DnaE intein. As chloroplast DNA is not transmitted by pollen in plants such as corn and soybean, transgene spread via pollen may be controlled in the future by expressing inactive gene fragments from separate DNA locations, such as the nuclear and chloroplast genome, and using the split intein to generate protein activity.     

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX , which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA . The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se .     

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.     

Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions

Inteins are selfish genetic elements that disrupt the sequence of protein-coding genes and are excised post-translationally. Most inteins also contain a HEN (homing endonuclease) domain, which is important for their horizontal transmission. The present review focuses on the evolution of inteins and their nested HENs, and highlights several unsolved questions that could benefit from molecular genetic approaches. Such approaches can be well carried out in halophilic archaea, which are naturally intein-rich and have highly developed genetic tools for their study. In particular, the fitness effects of harbouring an intein/HEN can be tested in direct competition assays, providing additional insights that will improve current evolutionary models.     

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.     

Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system

The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.     

DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0. 6) than in a DeltapolB (approximately 0.1) strain. Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.     

Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.