An in vivo screening system against protein splicing useful for the isolation of non-splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis

Lew, Belinda M.; Paulus, Henry

doi:10.1016/s0378-1119(01)00836-8

Cited by 32 publications

(29 citation statements)

References 26 publications

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“…Several groups have independently evolved new function into cis-splicing inteins (15,(25)(26)(27). However, directed protein evolution has not been applied to split inteins or, to our knowledge, any other protein complementation pair.…”

Section: Discussionmentioning

confidence: 99%

Traceless protein splicing utilizing evolved split inteins

Lockless

Muir

2009

Proc. Natl. Acad. Sci. U.S.A.

103

140

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Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cellbased selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects. directed evolution ͉ protein ligation ͉ Crk-II ͉ SCA ͉ coevolution P rotein function is modulated by a variety of posttranslational modifications, such as phosphorylation, ubiquitylation, and methylation (1). One of the most dramatic posttranslational modifications is protein splicing, an autocatalytic process in which an intervening polypeptide sequence, termed an intein, is excised from a precursor protein with concomitant splicing of the franking sequences, known as exteins. Protein splicing has proven to be a highly versatile process for methods development and forms the hub of several protein engineering technologies (2). Inteins have been successfully used in protein semisynthesis to create posttranslationally modified proteins (2, 3), in the cyclization of peptides to create small molecule toxins (4), in plant biotechnology to reconstruct proteins in vivo (5), in the segmental labeling of proteins for NMR studies (6), and in protein semisynthesis in living cells (7).Inteins come in 2 flavors-cis splicing inteins are single polypeptides that are embedded in a host protein, whereas trans-splicing inteins (herein called split inteins) are separate polypeptides that mediate protein splicing after the intein pieces and their protein cargo associate (8, 9) (Fig. 1A). Despite the many applications of split inteins in chemical biology and protein chemistry, they are plagued with various idiosyncratic parameters that limit their more general use. Most importantly, the 2 parts of the naturally split inteins associate and typically splice at a C-terminal junction containing the canonical ''CFN'' tripeptide sequence (10, 11), which are the first 3 amino acids of the C-extein sequence (Fig. 1 A). This tripeptide remains in the product after splicing, meaning that it will most often be a mutant protein. It is currently unclear why this "CFN" sequence is required for efficient protein trans-splicing. In contrast, work from several laboratories, incl...

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Section: Discussionmentioning

confidence: 99%

Traceless protein splicing utilizing evolved split inteins

Lockless

Muir

2009

Proc. Natl. Acad. Sci. U.S.A.

103

140

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“…To this end, rapid methods are desired to screen for compounds capable of blocking protein splicing. Three in vivo methods for detection of splicing and nonsplicing intein variants were recently published (1,3,11).This report describes a new genetic selection system for the identification of splicing and nonsplicing intein variants inserted into phage RB69 DNA polymerase. The method is based on growth versus lysis of Escherichia coli cells infected with conditionally defective T4 gp43 Ϫ phage, which contains amber mutations in the T4 DNA polymerase gene (gene 43) that render the phage inviable in nonsuppressor strains.…”

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confidence: 99%

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confidence: 99%

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

Cann

Amaya

Southworth

et al. 2004

Appl Environ Microbiol

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A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.Over the last decade, intensive research has yielded important insights into the mechanism by which biological activity is restored to proteins invaded by inteins (4,5,12,13). Unlike introns, which are removed from RNA before translation, inteins are cotranslated with the invaded protein to form a precursor polypeptide (4, 5, 13). The intein is then self-catalytically excised from the precursor, with concomitant ligation of the upstream (N extein) and downstream (C extein) flanking polypeptides (4, 5), to yield two proteins: the mature host protein and the intein. Inteins are often found in active sites and conserved motifs of proteins indispensable to cell metabolism (6,8,9). Inteins are therefore new antimicrobial targets in pathogens with inteins in essential proteins since splicing must occur for normal cell function. To this end, rapid methods are desired to screen for compounds capable of blocking protein splicing. Three in vivo methods for detection of splicing and nonsplicing intein variants were recently published (1,3,11).This report describes a new genetic selection system for the identification of splicing and nonsplicing intein variants inserted into phage RB69 DNA polymerase. The method is based on growth versus lysis of Escherichia coli cells infected with conditionally defective T4 gp43 Ϫ phage, which contains amber mutations in the T4 DNA polymerase gene (gene 43) that render the phage inviable in nonsuppressor strains. As a result, colony formation is observed with T4-susceptible E. coli strains lacking amber suppressors, such as ER2566. Plasmidborne DNA polymerase from the closely related phage RB69 can complement this defect in T4 gp43 Ϫ phage, resulting in cell lysis (10). This system for controlling phage viability was converted into a genetic selection system for protein splicing by in-frame insertion of an intein gene into the active site of the plasmid-encoded RB69 DNA polymerase gene (Fig. 1), rendering the RB69 DNA polymerase inactive in the absence of protein splicing. T4 gp43 Ϫ phage viability would then require protein splicing to produce active RB69 DNA polymerase (Fig. 2).The pol-2 Tli intein gene portion of Thermococcus litoralis (Vent) DNA polymerase (7) was cloned into the homologous active-site, region

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“…It was also used to screen for mutations or inhibitors that interfere with protein splicing mediated by the RecA intein of Mycobacterium tuberculosis. This screening procedure involved activation of the CcdB protein by protein splicing, such that host cells survive in the presence of inducer only when protein splicing was blocked (Lew and Paulus, 2002). CcdB is also used as a general selection marker in the gateway system (Invitrogen, CA).…”

Section: The Use Of Ccdb As a Reporter Genementioning

confidence: 99%

“…The single fusion that was reported was a lacZ N-terminal fusion to ccdB (Bernard, 1996;Gabant et al, 1997). Personal communications with Paulus H (Lew et al, 2002) (Personal communications, 2009) revealed that CcdB lost its lethal effect when 25 or more amino acid residues were fused to the N terminus. In this work we have presented the results of C terminal fusions.…”

Section: Ccdb Loss Of Function and Redsigning Of The Target Plasmidmentioning

confidence: 99%

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

AbdelRahman¹

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An in vivo screening system against protein splicing useful for the isolation of non-splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis

Cited by 32 publications

References 26 publications

Traceless protein splicing utilizing evolved split inteins

Traceless protein splicing utilizing evolved split inteins

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

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