Controllable intein splicing and N-terminal cleavage at mesophilic temperatures

Abstract

Inteins (intervening proteins) interrupt host proteins and are removed through a protein splicing reaction that ligates adjacent N- and C-exteins. The ability of inteins to specifically rearrange peptide bonds has proven exceptionally useful in protein engineering, thus, methods to control intein activity are of considerable interest. One particularly useful application of inteins is for the removal of an affinity tag following purification of a target protein through N-terminal cleavage (NTC). Typically, extended incubation at high temperature (greater than 50°C) or with an external nucleophile (e.g., dithiothreitol) is required to drive NTC, conditions that compromise the folding of many target proteins. Here, we characterize a variant of the Thermococcus kodakarensis RadA intein that can perform NTC at moderate temperatures in the absence of an external nucleophile. While we find that while NTC is largely inhibited during expression in Escherichia coli at 15°C, rapid and efficient NTC can be activated 37°C. Our results provide an alternative intein-based system - one that does not require either an external nucleophile or prolonged incubation at high temperature to stimulate NTC - that controls intein activity within a temperature range amenable to most mesophilic experimental organisms.

Keywords: N-terminal cleavage; biosensor; bioseparations; intein; protein splicing.

FIGURE 1

Mechanism of protein splicing and N-terminal cleavage: (A) Splicing and (B) NTC schemes with −1, 1, and +1 positions, exteins (black), and intein (gray) indicated. Relevant chemical groups essential to protein splicing and NTC are drawn. Details for each step are described in the main text.

FIGURE 2

TkΔE splicing and TkΔE-AA N-terminal cleavage (NTC) is activated by increasing temperature. (A) Schematic of MBP-Intein-GFP (MIG) reporter. MBP is colored gray, intein is colored red, and GFP is colored green. GFP-containing fluorescent products are visualized in-gel following semi-native PAGE. (B) Splicing of TkΔE in MIG reporter at different temperatures for 60 min. Precursor (M-I-G) and ligated exteins (M-G) bands are indicated. (C) Quantification of splicing at different temperatures from three independent splicing reactions. (D) MIG reporter with mutations to allow for NTC rather than splicing. Colored as in panel (A) (E) NTC TkΔE-AA in MIG reporter over 180 min at different temperatures. Precursor (M-I-G) and intein-GFP (I-G) products are indicated. (F) Quantification of splicing at different temperatures from three independent cleavage reactions. Error bars indicate standard deviation. When error bars are not shown in panels C and F, they are smaller than the symbol. Protein size markers are in kilodaltons.

FIGURE 3

TkΔE-AA NTC with 20 common amino acids in the -1 position. (A) Schematic of MBP-Intein-GFP (MIG) reporter with the −1 position indicated. Exteins and intein colored as in Figure 1. (B) Quantification of NTC with different residues at −1 position of TkΔE-AA after incubation for 2 (white bars), 6 (gray bars), and 20 (black bars) hours at 37°C. The relative rates of NTC for each −1 position, as described in the main text, are indicated with red (fast), green (moderate), or blue (slow). One letter amino acids abbreviations are shown. (C) Examples of relative NTC rates for a fast, moderate, and slow −1 residue. NTC after 2 h at 37°C for TkΔE-AA with -1H (fast), -1M (moderate), and -1I (slow). (D) TkΔE-AA-1D (Asp in −1 position) does not undergo premature NTC following expression at 15°C for 20 h and proceeds following incubation at 37°C for indicated times. Quantification in panel A is done as described in Figure 1. Protein size markers are in kilodaltons.

FIGURE 4

Homologous intein from Pyrococcus horikoshii is not prone to NTC and on-column NTC of the TkPl-AA variant. (A) TkPl-AA, but not Ph-AA, in the MIG reporter undergoes efficient NTC during incubation at 37°C for 16 h. (B) Quantification of NTC for TkPl-AA and Ph-AA following incubation at 37°C for 16 h (C) TkPl-AA MIG precursor intein can be purified, and, after incubation for 16 h at 37°C, two products are observed by Coomassie staining with sizes consistent with the cleaved MBP and intein-GFP. (D) TkP1-AA MIG precursor intein maintains NTC activity at 37°C while on beads following affinity resin capture and washing. In panel D, T indicates the product eluted from total amount of beads prior to incubation at 37°C, R is the product released into solution following incubation at 37°C, and U is the product that remained on the beads following incubation at 37°C. In all panels, T₀ is as described in Figure 1 and precursor (M-I-G), MBP (M) and intein-GFP (I-G) bands are indicated. Quantification and error are determined based on at least three independent NTC reactions as described in Figure 1. Protein size markers are in kilodaltons.