High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34–39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.


Introduction
Antimicrobial peptides (AMPs) normally consist of 12 to 50 amino acids and can be classified as α-helices [1], β-sheets [2], extended [3], and looped peptides [4,5]. Most AMPs exert their antimicrobial activities through the incorporation and permeabilization of microbial membranes, hence the death of microbial cells [6][7][8]. AMPs can work alone or in combination with antibiotics to diminish antibiotic-resistant pathogens and reduce the amount of antibiotics that are needed [9,10]. Recent progress of AMPs conjugated with antibiotics also demonstrated an enhanced killing effect on drug-resistant bacterial strains [11,12]. Moreover, many AMPs possess lipopolysaccharide (LPS) neutralization as well as anticancer activities [13][14][15]. Recent studies also summarized the immunomodulatory activities of AMPs in medical uses [15].
Cecropins are a family of AMPs that are widely found in the innate immune systems of Cecropia moths and are composed of 34 to 39 amino acids [16]. The structure of cecropins includes an N-terminal amphipathic α-helix, a hinge motif, and a hydrophobic C-terminal

Construction of the Expression Plasmid
Six histidines were attached in the N-terminus of intein, and the sequence of KR12AGPWR6 was attached to the C-terminus of intein ( Figure 1A). The amino acid sequences and optimized DNA sequences are shown in Figure 1B. The pET11b-His6-intein construct was synthesized from Protech Technology Enterprise Co., Ltd. (Taipei, Taiwan).

Expression of His6-Intein-KR12AGPWR6
The constructed expression vector was inserted into E. coli BL21 (DE3) c cells for expression. A single colony was selected and incubated in 100 mL LB taining ampicillin (0.1 mg/mL) at 37 °C with 150 rpm shaking overnight. A tota incubated cell culture was then inoculated into 1000 mL LB broth containing (0.1 mg/mL) until the OD600 reached 0.6 to 0.8. The protein was subsequently ind 0.4 mM IPTG for 24 h at 20 °C. After induction, the cells were harvested by cent at 6000× g for 20 min at 4 °C. The supernatant was discarded and the pellets were −20 °C.

Expression of His6-Intein-KR12AGPWR6
The constructed expression vector was inserted into E. coli BL21 (DE3) competent cells for expression. A single colony was selected and incubated in 100 mL LB broth containing ampicillin (0.1 mg/mL) at 37 • C with 150 rpm shaking overnight. A total of 25 mL incubated cell culture was then inoculated into 1000 mL LB broth containing ampicillin (0.1 mg/mL) until the OD 600 reached 0.6 to 0.8. The protein was subsequently induced with 0.4 mM IPTG for 24 h at 20 • C. After induction, the cells were harvested by centrifugation at 6000× g for 20 min at 4 • C. The supernatant was discarded and the pellets were stocked at −20 • C.

Purification of His6-Intein-KR12AGPWR6
The cell pellets were resuspended in lysis buffer (20 mM sodium phosphate, 150 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, pH 8.0) and lysed with a High-Pressure Homogenizer (AVESTIN EmulsiFlex C3, Mannheim, Germany). The cell lysates were separated with high-speed centrifugation at 4 • C (12,500× g, 30 min), and the supernatant was filtered with 0.2 µm filter before being applied to a Ni-NTA resin (QIAGEN, Hilden, Germany) equilibrated with lysis buffer. The target protein sample was separated under different concentrations of imidazole. The imidazole concentration was 40 mM at the washing steps and 400 mM at the elution steps, respectively. The eluted samples were concentrated using Amicon ® Stirred Cells with 3 kDa membrane (Merck Millipore, Burlington, MA, USA), and followed by dialysis using the cellulose tubular membrane (3.5 kDa MWCO, Cellu·Sep T1 Membrane, Membrane Filtration Products, Inc., Seguin, TX, USA) within 20 mM phosphate buffer (pH 8.0) at 4 • C for 24 h.

Intein Self-Cleavage
Self-cleavage of purified His6-Intein-KR12AGPWR6 was performed in high pH buffer (20 mM phosphate buffer, pH 10.0) at 55 • C for 72 h. After intein self-cleavage, the rKR12AGPWR6 was precipitated in the pellet. Then, the rKR12AGPWR6 peptide was obtained by high-speed centrifugation (12,500× g, 30 min, 4 • C), and the supernatant was discarded. The rKR12AGPWR6 peptide was further resuspended by 6 M guanidine hydrochloride before the purification by RP-HPLC [36].

Purification of rKR12AGPWR6 by RP-HPLC
All of the samples were purified by using C18 reversed-phase high-performance liquid chromatography (RP-HPLC) on the Prominence HPLC System (Shimadzu, Kyoto, Japan) and using a COSMOSIL C 18 -AR-II column (Nacalai Tesque, Kyoto, Japan). The column was equilibrated with ddH 2 O containing 0.1% (v/v) trifluoroacetic acid (TFA) and eluted with a gradient step from 15 to 100% (v/v) methanol containing 0.1% (v/v) TFA for 75 min at a flow rate of 1 mL/min. Signals were detected by UV 220 nm. The protein samples were collected and lyophilized, then resuspended in water, and the concentrations were determined by the bicinchoninic acid assay (BCA) method (GeneCopoeia TM , Rockville, MD, USA). The molecular mass of KR12AGPWR6 was verified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

Antimicrobial Activity Assays
Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Acinetobacter baumannii 14B0100, were purchased from Bioresources Collection & Research Center (BCRC, FIRDI, Hsinchu, Taiwan). The antibacterial activities of peptides were determined by the standard broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [37]. Briefly, the bacteria were incubated in MHB overnight at 37 • C. The cell cultures were regrown to mid-log phase and subsequently diluted to a final concentration of 5 × 10 5 CFU/mL. The peptides were loaded into each well at the final concentration of 64, 32, 16, 8, 4, 2, 1 µg/mL, then the microbes were loaded into each well of a polypropylene 96-well plate. After 16 h of incubation at 37 • C, the MIC values of peptides were determined by inspecting the visible growth of bacteria. The MIC values were defined as the lowest concentration of an antimicrobial agent that inhibits the visible growth of a microorganism. All experiments were repeated three times independently.

Circular Dichroism Spectroscopy
Circular dichroism spectra were recorded in the far-UV spectral region (190 to 260 nm) at 25 • C using a 0.1 cm path-length cuvette on an AVIV CD spectrometer (Aviv Biomedical Inc., Lakewood, NJ, USA). The peptide concentration was 60 µM in 20 mM sodium phosphate buffer or in 30% TFE buffer at pH 7.4.

Nuclear Magnetic Resonance Spectroscopy (NMR)
The NMR experiments were performed using 0.6 mM of 15 N-labeled samples. The NMR samples were prepared in NMR buffer (20 mM sodium phosphate buffer, 10 mM NaN 3 , pH 4.5) with 10% D 2 O (v/v) for field/frequency lock. 15 N-edited 2D HSQC (Heteronuclear Single-Quantum Correlation) spectroscopy; NOESY (Nuclear Overhauser Effect Spectroscopy); and TOCSY (Total Correlation Spectroscopy) experiments were recorded at 298 K on a Bruker Avance 600-MHz NMR spectrometer (Bruker, Billerica, MA, USA). 1 H NMR data were referenced to 1 H resonance frequency of DSS (2,2-dimethyl-2-siapentane-5sulfonate). Quadrature detection in the indirect dimensions was determined by using the States-TPPI (time-proportional phase incrementation) method. Signals from H 2 O were suppressed through low power presaturation (pulse program: zgpr). An analysis of the spectra was conducted using the Sparky software (T.D. Goddard and D.G. Kneller, SPARKY3, University of California, San Francisco, CA, USA).

Construction of the Recombinant Plasmid
The pET-11b plasmid was used as a template and the designed construct His6-intein-KR12AGPWR6 was subcloned into the expression vector ( Figure 1A). The N-terminal consecutive histidine served as purification tags. The amino acid sequence and the optimized DNA sequence were shown in Figure 1B. A DNA sequence analysis demonstrated that the His6-intein-KR12AGPWR6 sequence was correct.

Expression, Extraction and Purification of Recombinant His6-Intein-KR12AGPWR6
E. coli BL21 (DE3) cells containing the pET11b-His6-intein-KR12AGPWR6 plasmid were successfully induced by IPTG, and the expression of His6-Intein-KR12AGPWR6 was analyzed by SDS-PAGE ( Figure 2). As shown in Figure 2A, lanes 3 and 4 represented expression of His6-Intein-KR12AGPWR6 at 20 • C for 4, 8, and 24 h, respectively. The bands of His6-Intein-KR12AGPWR6 were observed, and the protein levels increased along with the induction time. As shown in Figure 2B, the relative intensity of lane 5 (24 h induction with IPTG) displayed the highest level of protein expression. The enhanced background impurities in lane 5 corresponding to 24 h induction with IPTG were removed by Ni-NTA purification ( Figure 3, lane 4-6). Lanes 6 to 8 showed protein expression levels without IPTG induction and smaller amounts of recombinant proteins were observed ( Figure 2A). Subsequently, the pellets were resuspended in lysis buffer and lysed with a high-pressure homogenizer. Ni-NTA resin was used for protein purification. As can be seen in Figure 3, the targeted proteins were examined in cell lysate (lane 2). No target proteins could be observed in the solution flow through (lane 3). The recombinant proteins were washed three times by 40 mM imidazole (lanes 4-6) to remove non-specific targets, and most of the nonspecific targets were washed out the first time. Intein may perform its self-cleavage activity at low salt concentrations. In our previous study, we proposed a 'prohibition condition' to inhibit the self-cleavage activity of intein before using Ni-NTA column to purify the target protein [38]. The 'prohibition condition' in this intein system includes a condition with salt concentration > 300 mM and pH < 7. Therefore, we chose 400 mM imidazole to prevent intein self-cleavage before the purification steps. The target proteins were then eluted by 400 mM imidazole (lane 7-10) and the protein band of recombinant His6-Intein-KR12AGPWR6 (MW 19.7 kDa) was observed on SDS-PAGE at about 20 kDa ( Figure 3). Imidazole was removed from the eluted sample using cellulose tubular membrane (3.5 kDa MWCO) in dialysis buffer at 4 • C for 24 h before intein self-cleavage.
Biomedicines 2022, 10, x FOR PEER REVIEW 6 of 14 ( Figure 3). Imidazole was removed from the eluted sample using cellulose tubular membrane (3.5 kDa MWCO) in dialysis buffer at 4 °C for 24 h before intein self-cleavage.

Optimization of Intein's Self-Cleavage
Reaction time, pH, and temperature conditions were used to optimize intein's selfcleavage. After intein's self-cleavage, the mixtures were analyzed by SDS-PAGE ( Figure  4A). Three major bands belonging to His6-intein-KR12AGPWR6, His6-intein, and KR12AGPWR6 were seen, and their molecular weights were 19.7 kDa, 16.9 kDa, and 2.8 kDa, respectively ( Figure 4A, lane 3-8). During intein self-cleavage, we observed that KR12AGPWR6 precipitated in the pellet. Thus, it is difficult to use the quantification of KR12AGPWR6 on SDS-PAGE to find the optimized intein self-cleavage condition. On the other hand, His-intein-KR12AGPWR6 and His6-intein dissolved in the supernatant. Therefore, we used the quantification of His-intein-KR12AGPWR6 and His6-intein on SDS-PAGE to determine the best self-cleavage conditions of intein. The band of KR12AG-PWR6 was the same as synthetic KR12AGPWR6 (MW 2.8 kDa) (lane 10), which suggested that KR12AGPWR6 was released via intein's self-cleavage. To optimize intein's self-cleavage efficacy, purified His6-intein-KR12AGPWR6 was kept in various pH buffers at 55 °C for 18 and 72 h. As shown in Figure 4A,B, the self-cleavage rate of intein at 72 h was higher than 18 h under different pH conditions. Further, a higher pH (pH = 10) exhibited better intein self-cleavage activity. Similarly, intein self-cleavage activity at 72 h was higher than Biomedicines 2022, 10, x FOR PEER REVIEW 6 of 14 ( Figure 3). Imidazole was removed from the eluted sample using cellulose tubular membrane (3.5 kDa MWCO) in dialysis buffer at 4 °C for 24 h before intein self-cleavage.

Optimization of Intein's Self-Cleavage
Reaction time, pH, and temperature conditions were used to optimize intein's selfcleavage. After intein's self-cleavage, the mixtures were analyzed by SDS-PAGE ( Figure  4A). Three major bands belonging to His6-intein-KR12AGPWR6, His6-intein, and KR12AGPWR6 were seen, and their molecular weights were 19.7 kDa, 16.9 kDa, and 2.8 kDa, respectively ( Figure 4A, lane 3-8). During intein self-cleavage, we observed that KR12AGPWR6 precipitated in the pellet. Thus, it is difficult to use the quantification of KR12AGPWR6 on SDS-PAGE to find the optimized intein self-cleavage condition. On the other hand, His-intein-KR12AGPWR6 and His6-intein dissolved in the supernatant. Therefore, we used the quantification of His-intein-KR12AGPWR6 and His6-intein on SDS-PAGE to determine the best self-cleavage conditions of intein. The band of KR12AG-PWR6 was the same as synthetic KR12AGPWR6 (MW 2.8 kDa) (lane 10), which suggested that KR12AGPWR6 was released via intein's self-cleavage. To optimize intein's self-cleavage efficacy, purified His6-intein-KR12AGPWR6 was kept in various pH buffers at 55 °C for 18 and 72 h. As shown in Figure 4A,B, the self-cleavage rate of intein at 72 h was higher than 18 h under different pH conditions. Further, a higher pH (pH = 10) exhibited better intein self-cleavage activity. Similarly, intein self-cleavage activity at 72 h was higher than

Optimization of Intein's Self-Cleavage
Reaction time, pH, and temperature conditions were used to optimize intein's self-cleavage. After intein's self-cleavage, the mixtures were analyzed by SDS-PAGE ( Figure 4A). Three major bands belonging to His6-intein-KR12AGPWR6, His6-intein, and KR12AGPWR6 were seen, and their molecular weights were 19.7 kDa, 16.9 kDa, and 2.8 kDa, respectively ( Figure 4A, lane 3-8). During intein self-cleavage, we observed that KR12AGPWR6 precipitated in the pellet. Thus, it is difficult to use the quantification of KR12AGPWR6 on SDS-PAGE to find the optimized intein self-cleavage condition. On the other hand, His-intein-KR12AGPWR6 and His6-intein dissolved in the supernatant. Therefore, we used the quantification of His-intein-KR12AGPWR6 and His6-intein on SDS-PAGE to determine the best self-cleavage conditions of intein. The band of KR12AGPWR6 was the same as synthetic KR12AGPWR6 (MW 2.8 kDa) (lane 10), which suggested that KR12AGPWR6 was released via intein's self-cleavage. To optimize intein's self-cleavage efficacy, purified His6-intein-KR12AGPWR6 was kept in various pH buffers at 55 • C for 18 and 72 h. As shown in Figure 4A,B, the self-cleavage rate of intein at 72 h was higher than 18 h under different pH conditions. Further, a higher pH (pH = 10) exhibited better intein self-cleavage activity. Similarly, intein self-cleavage activity at 72 h was higher than 18 h under different temperatures. The best intein cleavage condition for His6-Intein-KR12AGPWR6 was pH 10, 55 • C, for 72 h. Moreover, intein lost its self-cleavage activity at 4 • C, while the self-cleavage activity recovered at 37 • C. These results suggested that the most optimized conditions for the self-cleavage of His6-intein-KR12AGPWR6 were under high pH, high temperature, and longer reaction time.

Purification of KR12AGPWR6
KR12AGPWR6 was redissolved by using 6 M guanidine hydrochloride before R HPLC purification. Reversed-phase HPLC was used to purify KR12AGPWR6 with a gr dient of water/methanol containing 0.1% trifluoroacetic acid (TFA). The prolife of HPL chromatograms is displayed in Figure 5.

Purification of KR12AGPWR6
KR12AGPWR6 was redissolved by using 6 M guanidine hydrochloride before RP-HPLC purification. Reversed-phase HPLC was used to purify KR12AGPWR6 with a gradient of water/methanol containing 0.1% trifluoroacetic acid (TFA). The prolife of HPLC chromatograms is displayed in Figure 5.

Antimicrobial Activity
The antimicrobial activities of the recombinant rKR12AGPWR6 and chemically sy thesized sKR12AGPWR6 against S. aureus ATCC 25923, E. coli ATCC 25922, P. aerugino ATCC 27853, and A. baumannii BCRC 14B0100 were evaluated by MIC assay. As show in Table 1, the MIC value of rKR12AGPWR6 was the same as chemically synthesiz sKR12AGPWR6 against S. aureus ATCC 25,923 (2 µg/mL) and A. baumannii BCRC 14B01 (1 µg/mL). The recombinant rKR12AGPWR6 had a MIC of 4 µg/mL against E. c ATCC25922 and P. aeruginosa ATCC 27853. The results showed that rKR12AGPWR6 e

Antimicrobial Activity
The antimicrobial activities of the recombinant rKR12AGPWR6 and chemica thesized sKR12AGPWR6 against S. aureus ATCC 25923, E. coli ATCC 25922, P. ae ATCC 27853, and A. baumannii BCRC 14B0100 were evaluated by MIC assay. A in Table 1, the MIC value of rKR12AGPWR6 was the same as chemically syn sKR12AGPWR6 against S. aureus ATCC 25,923 (2 µg/mL) and A. baumannii BCRC (1 µg/mL). The recombinant rKR12AGPWR6 had a MIC of 4 µg/mL agains ATCC25922 and P. aeruginosa ATCC 27853. The results showed that rKR12AGPW

Antimicrobial Activity
The antimicrobial activities of the recombinant rKR12AGPWR6 and chemically synthesized sKR12AGPWR6 against S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and A. baumannii BCRC 14B0100 were evaluated by MIC assay. As shown in Table 1, the MIC value of rKR12AGPWR6 was the same as chemically synthesized sKR12AGPWR6 against S. aureus ATCC 25,923 (2 µg/mL) and A. baumannii BCRC 14B0100 (1 µg/mL). The recombinant rKR12AGPWR6 had a MIC of 4 µg/mL against E. coli ATCC25922 and P. aerug-inosa ATCC 27853. The results showed that rKR12AGPWR6 exhibited similar antimicrobial activities as the chemically synthesized sKR12AGPWR6 against both Gram-positive and Gram-negative bacteria.

Characterization of the Recombinant rKR12AGPWR6 by CD and NMR
CD spectroscopy was used to compare the structures of the recombinant rKR12A-GPWR6 and the chemically synthesized sKR12AGPWR6 (Figure 7). The CD results indicated that both the recombinant and chemically synthesized KR12AGPWR6 adopted a typical α-helical structure under 30% TFE buffer. In order to achieve backbone assignments of rKR12AGPWR6, the 2D TOCSY and NOESY spectra of KR12AGPWR6 in 20 mM phosphate buffer were obtained ( Figure S1). In addition, we successfully assigned the 1 H and 15 N backbone resonance peaks of rKR12AGPWR6 in buffer. A well-resolved 1 H-15 N HSQC spectrum of the fingerprint region of rKR12AGPWR6 is shown in Figure 8.

Characterization of the Recombinant rKR12AGPWR6 by CD and
CD spectroscopy was used to compare the structures of the PWR6 and the chemically synthesized sKR12AGPWR6 (Figure cated that both the recombinant and chemically synthesized KR typical α-helical structure under 30% TFE buffer. In order to ac ments of rKR12AGPWR6, the 2D TOCSY and NOESY spectra of K phosphate buffer were obtained ( Figure S1). In addition, we succ and 15 N backbone resonance peaks of rKR12AGPWR6 in buffer. HSQC spectrum of the fingerprint region of rKR12AGPWR6 is show
Recently, Malmsten and co-workers developed a strategy to increase the sa sistance of short antimicrobial peptides by adding tryptophan and/or phenylalanine tags [46][47][48]. End-tagging was also found to promote other biological effects, such as cancer and receptor binding activities [49,50]. We modified this strategy by addin naphthylalanine (Nal) to the termini of the short antimicrobial peptide S1 (Ac-KKW WLAKK-NH2) to boost its salt resistance, serum proteolytic stability, and antiendo activities [51,52]. We used solution NMR and paramagnetic relaxation enhancement niques to study the structural differences of S1 and S1-Nal-Nal in LPS micelles [53] three-dimensional structures of S1 and S1-Nal-Nal in complex with LPS clearly prov an explanation for the differences in their antiendotoxin activities. Based on these s tural results and the above-mentioned anti-LPS AMPs, we proposed a possible mod explain the mechanism of S1-Nal-Nal in the interaction with LPS. Firstly, S1-Na adopts a random coil structure in aqueous solution. Then, it is attracted to LPS by electrostatic interactions between the induced amphipathic helix and the negat charged region of LPS. Finally, the bulky hydrophobic β-naphthylalanine (Nal) end insert themselves into LPS by extra hydrophobic interactions with the lipid A regio LPS. The LPS-induced inflammation is then prohibited by the blocked lipid A region Based on the above-mentioned structural and functional studies of Cecropins an
Recently, Malmsten and co-workers developed a strategy to increase the salt resistance of short antimicrobial peptides by adding tryptophan and/or phenylalanine end-tags [46][47][48]. End-tagging was also found to promote other biological effects, such as anti-cancer and receptor binding activities [49,50]. We modified this strategy by adding β-naphthylalanine (Nal) to the termini of the short antimicrobial peptide S1 (Ac-KKWRKWLAKK-NH 2 ) to boost its salt resistance, serum proteolytic stability, and antiendotoxin activities [51,52]. We used solution NMR and paramagnetic relaxation enhancement techniques to study the structural differences of S1 and S1-Nal-Nal in LPS micelles [53]. The three-dimensional structures of S1 and S1-Nal-Nal in complex with LPS clearly provided an explanation for the differences in their antiendotoxin activities. Based on these structural results and the above-mentioned anti-LPS AMPs, we proposed a possible model to explain the mechanism of S1-Nal-Nal in the interaction with LPS. Firstly, S1-Nal-Nal adopts a random coil structure in aqueous solution. Then, it is attracted to LPS by the electrostatic interactions between the induced amphipathic helix and the negatively charged region of LPS. Finally, the bulky hydrophobic β-naphthylalanine (Nal) end-tags insert themselves into LPS by extra hydrophobic interactions with the lipid A region of LPS. The LPS-induced inflammation is then prohibited by the blocked lipid A region.
Based on the above-mentioned structural and functional studies of Cecropins and S1-Nal-Nal, we suspect that the binding and neutralization of LPS is not just through the sequences of Cecropins because some of the Cecropins and Cecropin analogues, although different in their sequences, can still bind to and neutralize LPS induced pro-inflammatory effects [18,40,46]. Therefore, we hypothesize that the binding and neutralization of LPS occurs through their specific structural features (i.e., amphipathic helix-AGP hinge-hydrophobic helix). Herein, we propose to extend this strategy to design AMPs with enhanced salt resistance and antiendotoxin activity. Some of the potential AMPs are listed in Table 2.

Expression and Purification of Cecropin-like AMPs
Intein is a protein segment that can cleave itself from a whole protein sequence and ligate the remaining N-terminal and C-terminal portions (the exteins) with a peptide bond [21]. Until now, over 600 inteins with different lengths have been identified. The specific cleavage-ligation function of intein has enabled various applications, such as protein engineering, isotope labeling, biomaterials, cyclization, and protein purification [54,55]. Recently, we have created an N-terminal mutated intein that has no N-terminal cleavage activity but preserves its C-terminal cleavage activity [21]. We have shown that this mutated intein can be used as a fusion tag and can self-cleave from the target protein. Moreover, we have shown that the efficiency of the intein self-cleavage is dependent on ionic strength, pH, temperature, and reaction time [21].
In this study, we used the mutated intein as a fusion partner for the expression and purification of KR12AGPWR6. We successfully expressed His6-intein-KR12AGPWR6 in E. coli ( Figure 2) and the recombinant His6-intein-KR12AGPWR6 was purified by Ni-NTA column ( Figure 3). In order to optimize the self-cleavage efficacy of His6-intein-KR12AGPWR6, the recombinant proteins were incubated at 55 • C under various pH buffers. The optimal pH for intein self-cleavage was 10. As can be seen in Table 1, the recombinant rKR12AGPWR6 possessed strong antimicrobial activities against both Gram-positive and Gram-negative bacteria, similar to the activities of the synthesized sKR12AGPWR6. We have also shown that the expressed rKR12AGPWR6 adopts an α-helical structure in TFE which is identical to the chemical synthesized sKR12AGPWR6. In addition, we demonstrated that the 15 N-labeled rKR12AGPWR6 that was produced in this study may be used to understand the structural features and interactions between rKR12AGPWR6 and model membranes/microbes for the design and development of useful antimicrobial peptides.
The lower yield of KR12AGPWR6 could be due to its antimicrobial activity, which makes itself potentially fatal to the expression host. However, the yield of KR12AGPWR6 is comparable to other antimicrobial peptides expressed by using the thioredoxin fusion or the GST fusion protein systems [56]. The premature intein self-cleavage before Ni-NTA column purification is also an intrinsic problem associated with the intein system. This problem causes loss in the yield of the target peptide. On the other hand, we could easily obtain the purified product by RP-HPLC by using this intein self-cleavage system. This intein self-cleavage system also requires no auxiliary enzymes or chemicals to remove the carrier protein. Additionally, by using bioreactors, volumetric protein production (E. coli cell density) could be improved up to 10-34 fold using a fed-batch strategy compared to batch cultivation [57]. Therefore, this intein self-cleavage system presents a potential method to produce AMPs in E. coli and is beneficial to reducing the cost of production for commercial scale production.
In addition to KR12AGPWR6, we chose CecropinA-AGP-WR6 (KWKLFKKIE-KVGQNIRDGIIK-AGP-RRWWRW) ( Table 2) to test this intein self-cleavage system. Figures S2 and S3 demonstrated that CecropinA-AGP-WR6 can be successfully obtained by using this intein self-cleavage expression and purification system. As shown in Table 3, CecropinA-AGP-WR6 also possesses strong antimicrobial activities against Gram-negative and Gram-positive bacteria. In conclusion, we successfully expressed the cecropin-like peptides rKR12AGPWR6 and CecropinA-AGP-WR6 from E. coli, and purification of rKR12AGPWR6 and CecropinA-AGP-WR6 was efficiently carried out by using the optimized intein self-cleavage system. This study provided a potential strategy to produce recombinant AMPs up to a commercial scale production, and this intein self-cleavage system can be widely applied to obtain other AMPs from E coli.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.