Intracellular Application of an Asparaginyl Endopeptidase for Producing Recombinant Head-to-Tail Cyclic Proteins

Abstract

Peptide backbone cyclization is commonly observed in nature and is increasingly applied to proteins and peptides to improve thermal and chemical stability and resistance to proteolytic enzymes and enhance biological activity. However, chemical synthesis of head-to-tail cyclic peptides and proteins is challenging, is often low yielding, and employs toxic and unsustainable reagents. Plant derived asparaginyl endopeptidases such as OaAEP1 have been employed to catalyze the head-to-tail cyclization of peptides in vitro, offering a safer and more sustainable alternative to chemical methods. However, while asparaginyl endopeptidases have been used in vitro and in native and transgenic plant species, they have never been used to generate recombinant cyclic proteins in live recombinant organisms outside of plants. Using dihydrofolate reductase as a proof of concept, we show that a truncated OaAEP1 variant C247A is functional in the Escherichia coli physiological environment and can therefore be coexpressed with a substrate protein to enable concomitant in situ cyclization. The bacterial system is ideal for cyclic protein production owing to the fast growth rate, durability, ease of use, and low cost. This streamlines cyclic protein production via a biocatalytic process with fast kinetics and minimal ligation scarring, while negating the need to purify the enzyme, substrate, and reaction mixtures individually. The resulting cyclic protein was characterized in vitro, demonstrating enhanced thermal stability compared to the corresponding linear protein without impacting enzyme activity. We anticipate this convenient method for generating cyclic peptides will have broad utility in a range of biochemical and chemical applications.

Figure 1

Direct production of cyclic mDHFR by recombinant gene expression. (A) Schematic coexpression system for generating cyclic mDHFR. The translated linear protein precursor to cyclic DHFR contains N- and C-terminal pro-peptides sequences (NTP and CTP, respectively) which are processed by OaAEP1 at the site indicated by black triangles. (B) Scheme of the proposed mechanism for mDHFR cyclization catalyzed by OaAEP1.

Figure 2

Mass analysis of cyclic and linear mDHFR. (A) SDS PAGE analysis of purified cyclic and linear mDHFR. (B) and (C) deconvoluted mass spectra of the purified cyclic and linear mDHFR demonstrates that upon cyclization 590 Da is lost due to loss of MRN from the N-terminus, GL from the C-terminus, and a water molecule during the condensation of N- and C-termini to form a native peptide bond.

Figure 3

Circular dichroism analysis of cyclic and linear mDHFR (10 μM). (A) CD spectra (20 °C) of mDHFR before and after thermal denaturation. (B) Thermal denaturation profile by monitoring the MRE at 209 nm indicating an increase in T_m of 3.7 °C for the H2T cyclic protein. Fraction of unfolded protein was calculated using eq 1 in the Supporting Information. T_m was estimated by converting the data to MRE and fitting to the Boltzmann sigmoidal equation using OriginPro graphing and analysis software. T_m is reported here as the mean average of triplicate experiments with the standard error of the mean.

Figure 4

Activity assay for cyclic mDHFR following the change in absorbance at 340 nm by NADPH during mDHFR catalyzed reaction. (A) Schematic of the mDHFR catalyzed reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. (B) UV/vis analysis monitoring the change in absorbance at 340 nm for turnover of NADPH at 20 °C (filled blue circles and red squares) showed comparable activity for both linear and cyclic versions. Following incubation at 45 °C for 15 min, the experiment was repeated at 20 °C and mDHFR activity again was monitored (open blue circles and red squares). Specific activity was calculated from the linear initial rate (first 2.5 min) using eq 2, Supporting Information. Data reported are averages with standard error of the mean from triplicate experiments.