Construction and Quantitation of a Selectable Protein Splicing Sensor Using Gibson Assembly and Spot Titers

Abstract

Inteins (intervening proteins) are translated within host proteins and removed through protein splicing. Conditional protein splicing (CPS), where the rate and accuracy of splicing are highly dependent on environmental cues, has emerged as a novel form of post‐translational regulation. While CPS has been demonstrated for several inteins in vitro, a comprehensive understanding of inteins requires tools to quantitatively monitor their activity within the cellular context. Here, we describe a method for construction of a splicing‐dependent system that can be used to quantitatively assay for conditions that modulate protein splicing. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Construction of an intein‐containing KanR2 library using Gibson assembly Basic Protocol 2: Phenotype determination using quantitative spot titers Support Protocol 1: Preparation of LB agar plates for spot titers Support Protocol 2: Preparation and transformation of competent M. smegmatis cells

Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria

“…4A ). M. smegmatis DnaBi1 splicing is strictly required for resistance, as mutation of the initiating nucleophile C-118 renders M. smegmatis cells sensitive to kanamycin ( 27 , 34 ) ( Fig. 4B ).…”

“…pACYC MIG DnaBi1, which expresses M. smegmatis DnaBi1 in the MIG reporter, and pACYC MIG Mle DnaBi, which expresses M. leprae DnaBi in the MIG reporter, were transformed into Escherichia coli BL21(DE3) cells for protein expression. pMBC-283 Kan R and pMBC-283 Kan R -DnaBi1WT were transformed into M. smegmatis MC 2 155 cells as described ( 34 ) for KISR survival assays described below.…”

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations

“…Somewhat surprisingly, when splicing active and inactive versions of the M. smegmatis DnaBi1 intein were inserted at 16 positions throughout the KanR protein, five displayed splicing-dependent resistance, eight displayed splicing-independent resistance, and three provided no resistance (Woods et al, 2020). Therefore, to ensure that kanamycin resistance cannot be provided by a KanR protein that is still interrupted by an intein, which would result in false positives when selecting for intein activity or potentially false negatives when searching for splicing inhibitors, a splicing inactive intein must also be tested at the same position within the KanR gene (Kelley et al, 2018;Woods et al, 2020;Woods et al, 2021). As expected, proximity to the active site correlated with the requirement of splicing for resistance, with all M. smegmatis DnaBi1-KanR splicing-dependent resistance fusions were within 14 residues and 16 Å of the active site (Woods et al, 2020).…”

“…Interrupted, inactive exteins are colored gray, inteins are colored pink if active and gray if inactivated by mutation, and active KanR is colored blue. hydrogen peroxide and zinc (Kelley et al, 2018;Woods et al, 2020;Woods et al, 2021), both stressors mycobacteria can encounter during challenge from the innate immune system (Zahrt and Deretic, 2002;Wagner et al, 2005;Neyrolles et al, 2015;Weiss and Schaible, 2015;Gao et al, 2018). Although M. smegmatis is generally non-pathogenic, and results may not translate exactly to mycobacteria that harbor conserved inteins like M. tuberculosis and M. leprae, it is useful as proxy for these pathogens.…”