Suppression of the toxicity of Bac7 (1–35), a bovine peptide antibiotic, and its production in E. coli
© Ishida and Inouye. 2016
Received: 29 December 2015
Accepted: 23 February 2016
Published: 2 March 2016
Bac7 (1–35) is an Arg- and Pro-rich peptide antibiotic, produced in bovine cells to protect them from microbial infection. It has been demonstrated to inhibit the protein synthesis in E. coli, leading to cell death. Because of its toxicity, no cost effective methods have been developed for Bac7 production in Escherichia coli for its potential clinical use. Here, we found a method to suppress Bac7 (1–35) toxicity in E. coli to establish its high expression system, in which Bac7 (1–35) was fused to the C-terminal end of protein S, a major spore-coat protein from Myxococcus xanthus, using a linker containing a Factor Xa cleavage site. The resulting His6-PrS2-Bac7 (1–35) (PrS2 is consisted of two N-terminal half domains of protein S connected in tandem) was well expressed using the Single-Protein Production (SPP) system at low temperature and subsequently purified in a single step by using a Ni column. The combination of protein S fusion and its expression in the SPP system at low temperature appeared to suppress Bac7 (1–35) toxicity. Both the purified His6-PrS2-Bac7 (1–35) and His6-PrS2-Bac7 (1–35) treated by Factor Xa were proven to be a potent inhibitor for cell-free protein synthesis.
Antimicrobial peptides (AMPs) consisting of 10–50 amino acid residues have been discovered from insects to mammals, specifically targeting against either Gram-negative bacteria or Gram-positive bacteria or both (Daher et al. 1988; Xi et al. 2014; Jayamani et al. 2015). Some of them are fungicidal. Some AMPs form helical structures through the membrane to cause cell lysis in a broad range of micro-organisms (Jamasbi et al. 2014; Li et al. 2015), while the others having very high contents of proline and arginine residues inhibit the functions of essential intracellular components (Tu et al. 2011; Roy et al. 2015). As recent emergence of highly antibiotic-resistant or multi-drug resistant pathogens, AMPs have become attractive alternatives for the treatment of patients. The use of AMPs is considered to be somehow less problematic than the use of conventional antibiotics as AMPs induce resistant strains in a much lower frequency (Zasloff 2002; Perron et al. 2006). Furthermore, the use of AMPs together with conventional antibiotics may have synergistic effects for therapeutic purpose. On the other hand, the disadvantages of AMPs are their cytotoxicity to the host, their instability in the cells, and the cost of their synthesis. Among AMPs, proline-rich antimicrobial peptides (PR-AMPs) have been isolated from mammalian neutrophils and from haemolymph of some invertebrate species (Anderson and Yu 2003; Treffers et al. 2005; Paulsen et al. 2013).
Bac7 (1–35) is a PR-AMP isolated from bovine and belongs to the cathelicidin family (Scocchi et al. 1997). The cathelicidins serve a critical role in mammalian innate immune defense against invasive bacterial infection (Zanetti 2004), and Bac7 (1–35) was found from bovine neutrophils together with cathelicidin as a antimicrobial peptides (Romeo et al. 1988; Gennaro et al. 1989). Later, Bac7 (1–35) was shown to inhibit DNA, RNA, and protein synthesis in E. coli (Mardirossian et al. 2014) after penetrating in the cells through SmbA, a peptide transporter (Mattiuzzo et al. 2007). Recently, Bac7 (1–35) was shown to bind to 70S ribosome resulting in inhibition of protein synthesis (Mardirossian et al. 2014). Despite the high toxicity to Gram-negative bacteria such as E. coli, Klebsiella pneumoniae, Salmonella typhimurium, and Enterobacter cloacae at 1–10 μM, Bac7 (1–35) has remarkably low cytotoxicity to the host mammalian cells (not toxic even at 50 μM) (Tomasinsig et al. 2006). Therefore, Bac7 (1–35) has been extensively studied because of its potential use for clinical application. A method to stabilize Bac7 (1–35) by PEGylation has been developed to reduce its renal clearance by which Bac7 (1–35) still retains its antibacterial activity as well as cell penetration activity (Benincasa et al. 2015).
For pharmaceutical applications, development of a method for an efficient AMP synthesis is important, but no successful expression system for Bac7 (1–35) has reported. So far, the production of some AMPs has been successfully carried out with a yeast system since these AMPs are not toxic to yeast (Jiménez et al. 2014; Wang et al. 2014; Mao et al. 2015). Furthermore, some AMPs have been produced using an E. coli system in combination with fusion tags such as thioredoxin (Feng et al. 2012) glutathione S-transferase (GST) (Feng et al. 2014), maltose-binding protein (MBP) (Velásquez et al. 2011) and small ubiquitin-like modifier like protein (SUMO) and subsequent cleavage of the AMPs form the fusion proteins by proteases such as thrombin, tobacco etch virus NIa protease, bovine coaglulation factor Xa, and enterokinase, which recognize only short, linear peptide sequences. However, if the AMP activity can be retained without cleavage from the fusion construct, it would be so much convenient for the toxicity assay of the AMPs. For this purpose, the SUMO technology has been successfully applied to many AMPs such as plectasin (Zhang et al. 2015), cathelicidin (Luan et al. 2014) and CM4 (Li et al. 2011).
In the present paper, we attempted to express Bac7 (1–35) in E. coli cells. Since Bac7 (1–35) is highly toxic to E. coli, it is essential to suppress its toxicity for its production. For this purpose, we tested two different protein tags, SUMO and protein S from Myxococcus xanthus to examine if the fusion tags could reduce the toxicity and enhance the expression of Bac7 (1–35). Protein S is a major spore-coat protein, which has been used as an effective fusion tag (Kobayashi et al. 2009). Protein S consists of 173 amino acids, which is composed of two homologous domains, the 92-residue N-terminal and the 81-residue C-terminal domains (Bagby et al. 1994). The expression vector, pCold-PST, contains two N-terminal domains (PrS2), repeated in tandem, to the C-terminal end of which a cloned protein or peptide is fused. pCold-PST vector has been shown to enhance the expression as well as the solubility of a cloned protein (Kobayashi et al. 2009). Protein S fused to a target protein has been shown not to severely affect the structure and function of the protein to be fused (Kobayashi et al. 2009, 2012). Since AMPs are toxic to the cells when expressed in E. coli, the suppression of toxicity to the cells possibly is essential for the production of AMPs. For this purpose, we attempted to use the Single-Protein Production (SPP) system for the production of PrS2-Bac7 (1–35), in which MazF, an ACA-specific endoribonuclease from E. coli is induced to eliminate almost all cellular mRNAs except for the mRNA for His6-PrS2-Bac7 (1–35) that is designed to have no ACA sequences without altering its amino acid sequence (Zhang et al. 2003; Suzuki et al. 2005, 2006). This enables us to produce only His6-PrS2-Bac7 (1–35) in E. coli cells without producing any other cellular proteins. Indeed, we were able to produce His6-PrS2-Bac7 (1–35) in a reasonable amount in E. coli cells, while with the SUMO tag, we were unable to express the protein.
Materials and methods
Construction of pColdPrS2Bac7 (1–35) vector and pColdSUMOBac7 (1–35)
A codon optimized ACA-less Bac7 (1–35) gene (cgtcgtattcgtccgcgtccaccgcgtctgccgcgtccgcgcccgcgtccactgccgttcccacgtccaggtccgcgtccgattccacgtccgctgccattcccgtaa) was synthesized (IDT) and cloned into ACA-less pColdPrS2 (Takara Bio) by using an infusion cloning system (Clontech), generating pColdPrS2-Bac7 (1–35), which is capable to produce His6-PrS2-Bac7 (1–35). PrS2 consists of two N-terminal half domains of protein S repeated in tandem (Kobayashi et al. 2009, 2012). His6-PrS2 and Bac7 (1–35) was linked with a tetra peptide, Ile-Glu-Gly-Arg as the Factor Xa cleavage site. Factor Xa cleaves the peptide after Arg so that intact Bac7 (1–35) is released after Factor Xa treatment without any extra amino acid residues attached.
The codon-optimized ACA-less SUMO-Bac7 (1–35) gene was synthesized (IDT) and cloned into pColdII (Takara Bio) by using infusion cloning system (Clontech), generating pColdSUMOBac7 (1–35) vector. In order to produce Bac7 (1–35) as a fusion protein, BL21(DE3) cells transformed with either pColdPrS2-Bac7 (1–35) or pColdSUMO-Bac7 (1–35) were inoculated into 10 ml of LB medium and the culture was incubated at 37 °C. When OD600 reached 0.8, the culture was transferred to 15 °C and the fusion proteins were induced by the addition of 1 mM IPTG. The mixture was further incubated for overnight.
Production and purification of His6-PrS2-Bac7 (1–35) using SPP system
BL21(DE3) co-transformed with pACYCmazF and pColdPrS2-Bac7 (1–35) was inoculated into 1 l of LB medium and the culture was incubated at 37 °C. When the OD600 reached 0.8, the culture was chilled on ice for 5 min, followed by incubation at 15 °C for another 1 h. Subsequently, 1 mM IPTG was added to induced MazF and the culture was incubated at 15 °C for overnight (Suzuki et al. 2006). The cells were collected by centrifugation and re-suspended into 20 ml of binding/washing buffer consisting of 20 mM Tris–HCl (pH 8.0), 500 mM NaCl, and 20 mM imidazole–HCl (pH 8.0). After breaking the cells by using a French press, the unbroken cells were removed by centrifugation at 14,000 rpm for 20 min. The supernatant thus obtained was subjected to further centrifugation at 50,000 rpm for 30 min to remove the membrane fraction. The supernatant fraction was mixed with 1 ml of Ni-resin equilibrated in binding/wash buffer and the mixture was incubated for 1 h at 4 °C. The Ni-resin was washed twice with 10 ml of washing buffer, and His6-PrS2-Bac7 (1–35) was eluted with 20 mM Tris–HCl (pH8), 500 mM NaCl and 300 mM imidazole–HCl (pH8.0). After collecting the eluted protein, the protein concentration was determined by the optical density at 280 nm using Nano Drop (Thermo Scientific), and the purity was examined by SDS-PAGE. After the protein fraction was dialyzed against 20 mM Tris–HCl (pH8.0) and 100 mM NaCl, it was concentrated to 3 mg/ml and stored at −80 °C.
Cleavage by factor Xa and identification of Bac7 (1–35)
The 30 μg of His6-PrS2-Bac7 (1–35) was digested with 4 μg of factor Xa in a 50 μl mixture containing 20 mM Tris–HCl (pH 8.0). 50 mM NaCl and 2 mM CaCl2. The reaction mixture was then incubated for 4 h at 37 °C, and the cleavage product was analyzed by 19 % SDS-PAGE followed by Coomassie blue staining. In order to confirm Bac7 (1–35) by mass spectrometry, His6-PrS2-Bac7 (1–35) was cleaved by factor Xa protease and the reaction mixture was diluted 25 times by the matrix solution containing sinapinic acid (10 mg/ml) in 0.1 % trifloroacetic acid and 50 % acetonitrile) and spotted on to a target plate (Opti-TOF 384 well insert, ABSciex) and air dried, followed by mass spectrometric analysis by a MALDI-TOF (4800 MALDI-TOF/TOF, ABSciex) using the positive mid-mass linear mode from 2 to 30 kDa.
Small scale purification of Bac7 (1–35) by ion-exchange column chromatography
Three hundred sixty μg of His6-PrS2-Bac7 (1–35) was digested with 5 μg of factor Xa in a 500-μl of 20 mM Tris–HCl (pH 8.0) containing 50 mM NaCl and 2 mM CaCl2. The reaction mixture was incubated for overnight at room temperature, and the cleaved Bac7 (1–35) was purified from the reaction mixture by ion-exchange chromatography using SP-Sepharose (GE healthcare). The column was equilibrated with 20 mM Tris–HCl (pH 8.0) and washed with 20 mM Tris–HCl (pH 8.0) containing 100 mM NaCl. Bac7 (1–35) was eluted with 20 mM Tris–HCl (pH 8.0) containing 1 M NaCl. All eluted fractions were collected and the Bac7 (1–35) concentration was determined at 595 nm with use of Pierce Coomassie Plus (Thermo Fisher Scientific) (Bradford 1976).
Synthesis of Bac7 (1–16)
Bac7 (1–16), the N-terminal fragment from residue 1 to residue 16 of Bac7 (1–35), consists of Arg–Arg-Ile-Arg-Pro-Arg-Pro–Pro-Arg-Leu-Pro-Arg-Pro-Arg-Pro-Arg, which has been shown to still retain one-fourth of the Bac7 toxicity and inhibit protein synthesis (Benincasa et al. 2004; Seefeldt et al. 2016). This peptide was commercially synthesized (GenScript) and dissolved in 1× PBS to make a 0.2 mM stock solution.
In vitro translation inhibition assay
PURExress In Vitro Protein Synthesis kit (New England BioLabs) was used in this study. The gene for dihydrofolate reductase (DHFR) was used as a positive control. The reaction mixture containing buffer A and buffer B supplied from NEB were mixed with 20 U of RNase inhibitor (Roche), linearlized DNA (10 ng/μl) and synthetic peptide Bac7 (1–16) (10 μM) or His6-PrS2-Bac7 (1–35) (10 μM) or water, and the reaction mixture was incubated for 2 h at 37 °C. Protein production was examined by SDS-PAGE followed by Coomassie blue staining.
Growth inhibition test when His6-PrS2-Bac7 (1–35) is induced in E. coli
The E. coli strain, BL21(DE3) harboring either pColdPrS2 or pColdPrS2-Bac7 (1–35) was grown in the M9-glucose medium. When the OD600 reached at 0.2, 1 mM IPTG was added into the medium to induce the protein. As a negative control, the culture medium in the absence of IPTG was also incubated and the OD600 was monitored every 30 min.
The antimicrobial activity of purified Bac7 (1–35) in E. coli
The E. coli strain BL21 (DE3) was grown in the M9-glucose medium and purified Bac7 (1–35) was added at the final concentration of 2 μM into the medium when OD600 reached 0.2. OD600 was monitored every 30 min.
The expression and purification of His6-PrS2-Bac7 (1–35)
After fractionation by ultracentrifuge, His6-PrS2-Bac7 (1–35) was fully recovered in the soluble fraction (Fig. 1b). The final yield after purification using Ni–NTA column chromatography was determined by a Nano Drop spectrophotometer to be 2.5 mg from 1 l LB medium. Higher than 90 % purification was achieved by one-step Ni–NTA purification (Fig. 1b).
Purification of Bac7 (1–35) from His6-PrS2-Bac7 (1–35)
After treating His6-PrS2-Bac7 (1–35) with factor Xa, cleaved Bac7 (1–35) was purified by ion-exchange column chromatography. Since the pI value of His6-PrS2 is 5.75 while the pI value of Bac7 (1–35) is 13.0, His6-PrS2 and Bac7 (1–35) were readily separated by SP Sepharose. In addition, the sizes of His6-PrS2 and Bac6(1–35) are 21 and 4.2 kDa, respectively, so that the size of Bac7 (1–35) is about one-sixth of the fusion protein. Fifty-four μg of highly purified Bac7 (1–35) was obtained from 360 μg of the fusion protein, which was about 90 % yield (Fig. 1d). Since 2.5 mg of His6-PrS2-Bac7 (1–35) was obtained from 1 l LB medium, the estimated yield of Bac7 (1–35) was 0.36 mg.
Identification of the Bac7 (1–35) fragment in His6-PrS2-Bac7 (1–35)
The function of His6-PrS2-Bac7 (1–35)
The antimicrobial activity of His6-PrS2-Bac7 (1–35) in the cells
Since His6-PrS2-Bac7 (1–35) was shown to retain the ribosome inhibition activity, we have tested the growth effect of induction of His6-PrS2-Bac7 (1–35) in the cells. As shown in Fig. 4a, the cell growth was totally arrested by His6-PrS2-Bac7 (1–35) after 30 min of induction, while the induction of His6-PrS2 did not cause the cell growth arrest.
The antimicrobial activity of purified Bac7 (1–35) using E. coli cells
The purified Bac7 (1–35) was tested using E. coli cells. Bac7 (1–35) efficiently inhibited E. coli cell growth at 2 μM after 30 min (Fig. 4b).
Bac7 (1–35) has been shown to inhibit the function of 70S ribosomes to block protein synthesis (Mardirossian et al. 2014). Thus, the activity of His6-PrS2-Bac7 (1–35) was tested using a cell-free protein expression system with a synthetic peptide Bac7 (1–16) as a protein synthesis inhibitor (Seefeldt et al. 2016) as a control. As shown Fig. 3b, the production of DHFR by the cell-free system was indeed inhibited by both His6-PrS2-Bac7 (1–35) and Bac7 (1–35) which was generated from His6-PrS2-Bac7 (1–35) by Factor Xa treatment which resulted in a small amount of uncleaved His6-PrS2-Bac7 (1–35) (Fig. 2c). Notably, the cleavage mixture effectively inhibited the protein synthesis (Fig. 3b). Since the minimum inhibitory concentration of Bac7 (1–35) has been reported to be 0.5 μM (Benincasa et al. 2004), it is assumed that there was an excessive amount of Bac7 (1–35) in the reaction mixture to inhibit protein synthesis. The PrS2 tag is known not to interfere with its fusion partner (Kobayashi et al. 2009); for example, PrS2 fused at the N-terminal end of OmpR, a phosphor sensory protein, did not inhibit the OmpR function at all (Kobayashi et al. 2009). Thus, it is not surprising to see that His6-PrS2-Bac7 (1–35) possesses an antibacterial activity in spite of the fact that the N-terminal part of Bac7 (1–35) has been shown to be crucial for the antimicrobial activity (Guida et al. 2015).
Using the SPP system, MazF cleaves at all ACA in mRNA while only the codon-optimized ACA-less gene for His6-PrS2-Bac7 (1–35) remains intact. Therefore, upon induction of MazF, only His6-PrS2-Bac7 (1–35) is produced in the cells (Fig. 5). Notably, in the SPP system, all the cellular mRNAs containing ACA sequences are digested by MazF, so that cell growth is completely arrested allowing the production of only the target protein from the ACA-less mRNA in the growth-arrested cells. In this manner, toxic proteins can still be produced as far as they do not inhibit ATP production and protein synthesis. Previously, we have demonstrated that it is possible to completely replace all arginine residues in a protein with canavanine, a highly toxic analogue of arginine using the SPP system, since the incorporation of canavanine into any other cellular proteins is well suppressed (Suzuki et al. 2006; Mao et al. 2009; Ishida et al. 2013). In the present paper, we combine both PST and SPP technologies (PST-SPP technology) to successfully express His6-PrS2-Bac7 (1–35).
In this study, we demonstrated to obtain 90 % pure His6-PrS2-Bac7 (1–35) by one-step purification. In addition, Bac7 (1–35) was readily purified from His6-PrS2-Bac7 (1–35) treated by factor Xa followed by ion exchange column chromatography using SP-Sepharose, since the pI value of His6-PrS2 is 5.75 while that of Bac7 (1–35) is 13.0. We were able to obtain highly pure Bac7 (1–35) with approximately 90 % yield. It is also important to note that since Bac7 (1–35) does not have any aromatic residues, the protein concentration should be determined by ninhydrin or the Bradford assay (Bradford 1976). While the chemical synthesis of long AMPs such as Bac7 (1–35) is highly expensive, the technology developed in the present paper will greatly reduce the cost of the AMP production.
YI and MI conceived of this study and wrote the manuscript. YI carried out the experiment. All authors read and approved the final manuscript.
This work is partially supported by a grant from the National Institutes of Health R01GM085449.
The authors have declared that they have no competing interests.
This article does not contain any studies with animals or human participants performed by any of the authors.
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- Anderson RC, Yu PL. Isolation and characterisation of proline/arginine-rich cathelicidin peptides from ovine neutrophils. Biochem Biophys Res Commun. 2003;312:1139–46.View ArticlePubMedGoogle Scholar
- Bagby S, Harvey TS, Eagle SG, Inouye S, Ikura M. NMR-derived three-dimensional solution structure of protein S complexed with calcium. Structure. 1994;2:107–22.View ArticlePubMedGoogle Scholar
- Benincasa M, Scocchi M, Podda E, Skerlavaj B, Dolzani L, Gennaro R. Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates. Peptides. 2004;25:2055–61.View ArticlePubMedGoogle Scholar
- Benincasa M, Zahariev S, Pelillo C, Milan A, Gennaro R, Scocchi M. PEGylation of the peptide Bac7 (1–35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity. Eur J Med Chem. 2015;95:210–9.View ArticlePubMedGoogle Scholar
- Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.View ArticlePubMedGoogle Scholar
- Daher KA, Lehrer RI, Ganz T, Kronenberg M. Isolation and characterization of human defensin cDNA clones. Proc Natl Acad Sci. 1988;85:7327–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Feng X, Liu C, Guo J, Song X, Li J, Xu W, Li Z. Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33. Appl Microbiol Biotechnol. 2012;95:1191–8.View ArticlePubMedGoogle Scholar
- Feng XJ, Xing LW, Liu D, Song XY, Liu CL, Li J, Xu WS, Li ZQ. Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli. J Ind Microbiol Biotechnol. 2014;41:527–34.View ArticlePubMedGoogle Scholar
- Gennaro R, Skerlavaj B, Romeo D. Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils. Infect Immun. 1989;57:3142–6.PubMedPubMed CentralGoogle Scholar
- Guida F, Benincasa M, Zahariev S, Scocchi M, Berti F, Gennaro R, Tossi A. Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7. J Med Chem. 2015;58:1195–204.View ArticlePubMedGoogle Scholar
- Ishida Y, Park JH, Mao L, Yamaguchi Y, Inouye M. Replacement of all arginine residues with canavanine in MazF-bs mRNA interferase changes its specificity. J Biol Chem. 2013;288:7564–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Jamasbi E, Batinovic S, Sharples RA, Sani MA, Robins-Browne RM, Wade JD, Separovic F, Hossain MA. Melittin peptides exhibit different activity on different cells and model membranes. Amino Acids. 2014;46:2759–66.View ArticlePubMedGoogle Scholar
- Jayamani E, Rajamuthiah R, Larkins-Ford J, Fuchs BB, Conery AL, Vilcinskas A, Ausubel FM, Mylonakis E. Insect-derived cecropins display activity against Acinetobacter baumannii in a whole-animal high-throughput Caenorhabditis elegans model. Antimicrob Agents Chemother. 2015;59:1728–37.View ArticlePubMedPubMed CentralGoogle Scholar
- Jiménez JJ, Borrero J, Gútiez L, Arbulu S, Herranz C, Cintas LM, Hernández PE. Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis. Mol Biotechnol. 2014;56:571–83.View ArticlePubMedGoogle Scholar
- Kobayashi H, Swapna GV, Wu KP, Afinogenova Y, Conover K, Mao B, Montelione GT, Inouye M. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility. J Biomol NMR. 2012;52:303–13.View ArticlePubMedPubMed CentralGoogle Scholar
- Kobayashi H, Yoshida T, Inouye M. Significant enhanced expression and solubility of human proteins in Escherichia coli by fusion with protein S from Myxococcus xanthus. Appl Environ Microbiol. 2009;75:5356–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Li JF, Zhang J, Zhang Z, Kang CT, Zhang SQ. SUMO mediating fusion expression of antimicrobial peptide CM4 from two joined genes in Escherichia coli. Curr Microbiol. 2011;62:296–300.View ArticlePubMedGoogle Scholar
- Li Y, Wu H, Teng P, Bai G, Lin X, Zuo X, Cao C, Cai J. Helical antimicrobial sulfono-γ-aapeptides. J Med Chem. 2015;58:4802–11.View ArticlePubMedGoogle Scholar
- Luan C, Zhang HW, Song DG, Xie YG, Feng J, Wang YZ. Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol. 2014;98:3651–8.View ArticlePubMedGoogle Scholar
- Mao L, Tang Y, Vaiphei ST, Shimazu T, Kim SG, Mani R, Fakhoury E, White E, Montelione GT, Inouye M. Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system. J Struct Funct Genomics. 2009;10:281–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Mao R, Teng D, Wang X, Zhang Y, Jiao J, Cao X, Wang J. Optimization of expression conditions for a novel NZ2114-derived antimicrobial peptide-MP1102 under the control of the GAP promoter in Pichia pastoris X-33. BMC Microbiol. 2015;15:57.View ArticlePubMedPubMed CentralGoogle Scholar
- Mardirossian M, Grzela R, Giglione C, Meinnel T, Gennaro R, Mergaert P, Scocchi M. The host antimicrobial peptide Bac (71–35) binds to bacterial ribosomal proteins and inhibits protein synthesis. Chem Biol. 2014;21:1639–47.View ArticlePubMedGoogle Scholar
- Mattiuzzo M, Bandiera A, Gennaro R, Benincasa M, Pacor S, Antcheva N, Scocchi M. Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides. Mol Microbiol. 2007;66:151–63.View ArticlePubMedGoogle Scholar
- Paulsen VS, Blencke HM, Benincasa M, Haug T, Eksteen JJ, Styrvold OB, Scocchi M, Stensvåg K. Structure-activity relationships of the antimicrobial peptide arasin 1—and mode of action studies of the N-terminal, proline-rich region. PLoS One. 2013;8:e53326.View ArticlePubMedPubMed CentralGoogle Scholar
- Perron GG, Zasloff M, Bell G. Experimental evolution of resistance to an antimicrobial peptide. Proc Biol Sci. 2006;273:251–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Romeo D, Skerlavaj B, Bolognesi M, Gennaro R. Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils. J Biol Chem. 1988;263:9573–5.PubMedGoogle Scholar
- Roy RN, Lomakin IB, Gagnon MG, Steitz TA. The mechanism of inhibition of protein synthesis by the proline-rich peptide oncocin. Nat Struct Mol Biol. 2015;22:466–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Scocchi M, Wang S, Zanetti M. Structural organization of the bovine cathelicidin gene family and identification of a novel member. FEBS Lett. 1997;417:311–5.View ArticlePubMedGoogle Scholar
- Seefeldt AC, Graf M, Pérébaskine N, Nguyen F, Arenz S, Mardirossian M, Scocchi M, Wilson DN, Innis CA. Structure of the mammalian antimicrobial peptide Bac7 (1-16) bound within the exit tunnel of a bacterial ribosome. Nucleic Acids Res. 2016.Google Scholar
- Suzuki M, Roy R, Zheng H, Woychik N, Inouye M. Bacterial bioreactors for high yield production of recombinant protein. J Biol Chem. 2006;281:37559–65.View ArticlePubMedGoogle Scholar
- Suzuki M, Zhang J, Liu M, Woychik NA, Inouye M. Single protein production in living cells facilitated by an mRNA interferase. Mol Cell. 2005;18:253–61.View ArticlePubMedGoogle Scholar
- Tomasinsig L, Skerlavaj B, Papo N, Giabbai B, Shai Y, Zanetti M. Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells. J Biol Chem. 2006;281:383–91.View ArticlePubMedGoogle Scholar
- Treffers C, Chen L, Anderson RC, Yu PL. Isolation and characterisation of antimicrobial peptides from deer neutrophils. Int J Antimicrob Agents. 2005;26:165–9.View ArticlePubMedGoogle Scholar
- Tu YH, Ho YH, Chuang YC, Chen PC, Chen CS. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip. PLoS One. 2011;6:e28197.View ArticlePubMedPubMed CentralGoogle Scholar
- Velásquez JE, Zhang X, van der Donk WA. Biosynthesis of the antimicrobial peptide epilancin 15× and its N-terminal lactate. Chem Biol. 2011;18:857–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang XJ, Wang XM, Teng D, Zhang Y, Mao RY, Wang JH. Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner. Lett Appl Microbiol. 2014;59:71–8.View ArticlePubMedGoogle Scholar
- Xi D, Wang X, Teng D, Mao R. Mechanism of action of the tri-hybrid antimicrobial peptide LHP7 from lactoferricin, HP and plectasin on Staphylococcus aureus. Biometals. 2014;27:957–68.View ArticlePubMedGoogle Scholar
- Zanetti M. Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol. 2004;75:39–48.View ArticlePubMedGoogle Scholar
- Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002;415:389–95.View ArticlePubMedGoogle Scholar
- Zhang C, He X, Gu Y, Zhou H, Cao J, Gao Q. Recombinant scorpine produced using SUMO fusion partner in Escherichia coli has the activities against clinically isolated bacteria and inhibits the Plasmodium falciparum parasitemia in vitro. PLoS One. 2014;9:e103456.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang L, Li X, Wei D, Wang J, Shan A, Li Z. Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector. J Ind Microbiol Biotechnol. 2015;42:1369–76.View ArticlePubMedGoogle Scholar
- Zhang Y, Zhang J, Hoeflich KP, Ikura M, Qing G, Inouye M. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Mol Cell. 2003;12:913–23.View ArticlePubMedGoogle Scholar