Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

Abstract

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

Recent progress in intein research: from mechanism to directed evolution and applications

“…Perler and coworkers, for example, have used molecular evolution in combination with positive selection systems to turn both the Mxe GyrA mini-intein as well as the Tli Pol-2 intein into temperature-sensitive forms as a potential system for growth-based screenings of protein splicing inhibitors [73,74]. Perrimon and colleagues have evolved the Sce VMA1 intein into temperature-sensitive derivatives to enable control of protein activity by temperature-regulated protein splicing in eukaryotes [75].…”

An Evolved Mxe GyrA Intein for Enhanced Production of Fusion Proteins

“…However, surface display levels of the scFvs were reduced by ∼40% when fused to intein compared to the unfused antibody. In addition, surface display of heterologous proteins is not ideally suited for protein production at a preparative scale since protein expression on the yeast surface is limited to ∼100 000 display constructs per yeast, , producing on the order of 70 μg of scFv per liter of yeast culture, whereas baseline scFv secretion in yeast is in the multimilligram per liter range. , The yeast display levels of scFv-intein proteins could potentially be improved via directed evolution, as has been previously reported for a variety of proteins. , Moreover, improvements in yeast display often translate to improvements in secretion titer. − While directed evolution approaches have been employed to engineer catalytic properties of inteins such as temperature, pH, and ligand dependence, − intein-fusion protein expression levels have not been a target for improvement.…”

Identification of theMycobacterium tuberculosisSUF Machinery as the Exclusive Mycobacterial System of [Fe-S] Cluster Assembly: Evidence for Its Implication in the Pathogen's Survival

“…Effectively, inteins are proteins embedded in-frame in a host protein; they are autocatalytically and posttranscriptionally excised from the peptide precursor to produce the functional host protein (26). Hence, the impediment of the protein splicing of a mycobacterial protein involved in a critical cellular process could represent an unusual way to kill M. tuberculosis (4,8,33).…”

“…Effectively, inteins are proteins embedded in-frame in a host protein; they are autocatalytically and posttranscriptionally excised from the peptide precursor to produce the functional host protein (26). Hence, the impediment of the protein splicing of a mycobacterial protein involved in a critical cellular process could represent an unusual way to kill M. tuberculosis (4,8,33).Among the intein-containing proteins, the mycobacterial RecA recombinase, while directly implied in DNA repair and homologous recombination (15, 31, 43), is not essential for the survival of Mycobacterium bovis BCG in a mouse infection model (43). An essential role of the M. tuberculosis DnaB helicase is more likely, based on the fact that DnaB is an essential component of the chromosome replication process in the pathogen Helicobacter pylori, as in Escherichia coli (42,47).…”