The heterologous expression strategies of antimicrobial peptides in microbial systems

Protein Expression and Purification 140 (2017) 52e59

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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep

The heterologous expression strategies of antimicrobial peptides in microbial systems Ting Deng a, 1, Haoran Ge a, 1, Huahua He a, Yao Liu a, Chao Zhai a, Liang Feng b, Li Yi a, * a

Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China b School of Environmental Studies, China University of Geosciences, 430074, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 July 2017 Received in revised form 8 August 2017 Accepted 8 August 2017 Available online 12 August 2017

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. © 2017 Elsevier Inc. All rights reserved.

Keywords: Heterologous expression Antimicrobial peptides Expression strategy

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Expression strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2.1. Codon optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2.2. Host strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2.3. Promoters using in host cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2.4. Tandem multimeric expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2.5. Fusion tag expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2.6. Hybridization expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

1. Introduction

* Corresponding author. Departmental of Microbiology, Departmental of Bioengineering, College of Life Science, Hubei University, Wuhan, Hubei, 430062, China E-mail address: [email protected] (L. Yi). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.pep.2017.08.003 1046-5928/© 2017 Elsevier Inc. All rights reserved.

AMPs are small molecular peptides with antimicrobial activities produced by many species [1,2]. Usually, AMPs consist of 6e60 amino acids, presenting strong alkaline and thermal stability, and broad antimicrobial spectrum. According to the Antimicrobial Peptide Database (APD) at the University of Nebraska Medical

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53

Fig. 1. Classification of AMPs. A: AMPs have been identified ranging from archaea to animals; B: Classification of antimicrobial activity of AMPs with resolved structures in CAMP database.

Center (http://aps.unmc.edu/AP/main.php/), more than 2700 AMPs have been identified so far, ranging from archaea to animal (Fig. 1A) [3]. In addition, the most updated Collection of Antimicrobial Peptide (CAMP, http://www.camp.bicnirrh.res.in/) database contains 757 high-resolution AMP structures (Fig. 1B), among which more than half are antibacterial peptides [4]. Typically, AMPs are rich of positively charged and hydrophobic residues (Fig. 2, Table 1). Due to their different characteristics, various classification methods of AMPs were proposed. For example, AMPs can be classified by their sources, including bacteria, archaea, fungi, plants, and animals. Another way to classify AMPs is based on their secondary structures, including a-helix, bsheet, extended structures, and loop structures [5]. How a short peptide could cause an extraordinary antimicrobial effect is always an interesting topic in biomedical research. Among many mechanisms that have been reported, two of them are currently widely acknowledged: the membrane disruptive mechanism and the non-membrane disruptive mechanism. In the membrane disruptive mechanism, AMPs are mostly amphipathic, mainly conferring ionic and hydrophobic interactions with membrane proteins. For example, cationic AMPs, such as Asprotegrin I and Magainin 2, can combine with anionic cell membrane via electrostatic force to make the cell membrane form unstable regions, where the AMPs are inserted into the lipid bilayer of the cell membrane to result in cell death [6]. This membrane disruptive mechanism includes Barrel theory [7] and Carpet model [8]. For the Barrel theory, AMPs are polymerized to form ion channels in the cell membrane, leading to the cell leakage. Comparably, the Carpet model states that AMPs can change the mobility and thickness of the cell membrane by binding to phospholipid molecules on the membrane, resulting in transient voids on the cell surface. In the non-membrane disruptive mechanism, AMPs quickly pass through the membrane without causing membrane damage. However, these AMPs can interact with the intracellular molecules to affect the synthesis and repair of DNA, the synthesis of protein, intracellular metabolism, or cell signal transduction to cause cell death [9]. For example, indolicidin covalently binds to DNA to prevent the DNA replication, which thereby impairs the cell division [10]. As therapeutic reagents, AMPs have been found to function on tumor inhibition and a variety of bacteria, viruses, and protozoa. The most attractive advantage of AMPs is that AMPs can specifically target on the prokaryotic cells or pathogen infected eukaryotic

cells, with almost no side-effect on healthy eukaryotic cells [11,12]. Biochemical analysis indicated that this special property of AMPs was mainly caused by the different cell surface compositions between bacterial and mammalian cells. The bacterial surface is characterized by its negatively charged environment with lipopolysaccharides and lipoteichoic acids, while mammalian cell surface contains zwitterionic phospholipids, cholesterol and sphingomyelin. Besides the specificity against microbes, another notable feature of AMPs is their low drug resistance. Unlike most antibiotics, AMPs usually act on non-specific targets, such as membrane proteins, which makes it difficult for the target microbes to develop drug resistance. Due to these advantages, AMPs have been widely applied in food engineering [13], medicine treatment [14,15], medical devices [16,17], agriculture [18], and so on. In order to better carry out their antimicrobial functions, AMPs with high purity are preferred to minimize the impurity caused side effects, especially in the clinical treatments. However, the simple structure and small molecular weight of AMPs lead to the challenge of obtaining purified AMPs with high biological activity in an efficient manner. In practical industries, three strategies, including direct isolation from natural sources, chemical synthesis, and heterologous expression, were the major methods currently applied, among which heterologous expression caught the most attention because of its low cost and high efficiency. Extraction from living organisms processes the advantage of obtaining AMPs with high biological activities, such as the lycotoxins extracted from the spider Lycosa carolinensis [19] and the peptide strongylocin from Strongylocentrotus droebachiensis [20]. However, the major issue for this method is that the extraction process is complicated with high cost, usually causing a low yield and impurity of the AMP products, even though highly active products can normally be obtained. Chemical synthesis is another way to produce AMPs, which can obtain AMP products with high purity. Nevertheless, the high cost during the synthesis process significantly limits its application, especially for the AMPs with long amino acid length. Compared to these two strategies, heterologous expression of AMPs has great advantages of low production cost, short production period, simple extraction process, and easily obtained high yields. In fact, different heterologous expression systems have been developed with distinct advantages for the expression of certain AMPs (Table 2). In here, the current progress of strategies utilized in different heterologous expression systems,

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T. Deng et al. / Protein Expression and Purification 140 (2017) 52e59

Fig. 2. The typical structures of AMPs. A: a-helical peptides; B: b-sheet peptides; C: extended peptides; D: loop peptides. The positively charged residues are marked in red, the hydrophobic residues are marked in gold, and the rest residues are marked in gray. All the structure files were extracted from PDB database (http://www.rcsb.org/pdb/home/home. do).

Table 1 Summary of different AMPs. Structure Mechanism

Example

Target/mode of action

Ref.

a-helix

Magainin II

Cell membrane permeabilization induction of apoptosis-like death LPS permeabilization Membrane thinning/thickening LPS-assembly protein D (LptD) inhibition Non-lytic membrane depolarization

[59]

Forming the toroidal pores composed of loosely associated peptides to disrupt bacterial membranes

Cecropin Ll-37 b-sheet Disrupting bacterial membranes via the formation of toroidal pores; also use a non-dissolving Protegrin I mechanism to bind to DNA or proteins in cells Bovine lactoferricin Plectasin Extended Containing high proportions of certain amino acids, specifically for Arg, Pro or Thr residues, Indolicidin which can cause membrane leakage or interact with intracellular proteins such as the heatshock protein Dnak and G- roEL Pyrrhocoricin

Loop

Interacting selectively with the negatively charged lipids, such as LPS

Inhibition of cell wall synthesis Cell membrane depolarization and lysis, inhibition of DNA synthesis Inhibition of protein synthesis, binding Dnak and GroEL Apidecin Inhibition of protein synthesis, binding to DnaK, GroEL and ribosomal proteins Tachyplesin I Cell membrane permeabilization Defensins Interaction with DNA in vitro Lipid II

[60] [61] [62] [63] [64] [65] [66] [67] [68] [69]

T. Deng et al. / Protein Expression and Purification 140 (2017) 52e59

55

Table 2 Main characteristics of expression systems utilized for exemplary AMPs' production. Expression system

Mainly promoter

AMP

Vector

Promoter

Signal peptide

Expression

Yield (mg/L)

Ref.

E.coli

Inducible

Ompa PhoA PelB SacB SacB a-MF

Fusion protein

Inducible Constitutive Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Constitutive Constitutive Constitutive Constitutive Constitutive Inducible

pET pHUE pSUMO pDM030 pGJ pPIC9k pICZaA pPICZaA pHILa pPICZaA pPIC9K pPIC9K pPICZaA pPICZaA pPICZB pPICZaA pPICZaA pPIC9K pPICZaA pGAPZ pGAPZaA pGAP pVT103L pPRL2 pYES2

T7

B. bacillus

Stomox-ynZH1 AN5-1 ABP-dHC-cecA CAM-W Cecropin AD SN-1 PaDef MP1106 LFcinC hepcidin-25 Abaecin Psc-AFP Protegrin-1 b-defensin2 CecP4 NZ17074 Fowlicidin-2 Ps-BD2 NZ2114 Plectasin cecropin D MP1102 Hbd-1 plantaricin 423 L50A and L50B

a-MF MFa1S MFa1S

Secretion

185 3.3 287.9 159 30.6 40 79.6 831 ND 1.9 ND 500 40 383.7 ND 4.1 85.6 ND 2390 370 485.2 807.4 0.55 56 0.56

[70] [71] [40] [33] [35] [72] [24] [73] [74] [75] [76] [76] [77] [78] [79] [54] [80] [81] [38] [82] [83] [37] [47] [46] [30]

P. pastoris

S.cerevisiae

Glv P43 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 AOX1 GAP GAP GAP ADH1 ADH1 GAL1

Secretion Secretion Secretion

ND: not determined.

Fig. 3. The summarized heterologous expression strategies of AMPs. (A) Codon optimization, (B) Tandem multimeric expression, (C) Fusion tag expression, (D) Hybridization expression: AMP-a and AMP-b combined together represent different types of AMPs.

including the E. coli, bacillus, and yeast, will be mainly introduced, which might provide guidance for the future development of AMP production (Fig. 3).

hbd2 was nine times higher with codon optimization, reaching a final concentration of 130 mg/L [21]. Similar results were also obtained with hbd5 and hbd6 in E. coli BL21 (DE3), whose expression levels reached 149 mg/L and 157 mg/L, respectively [22]. The obtained hbd5 and hbd6 AMPs showed antimicrobial activity against E. coli K12 but not Staphylococcus aureus, which was suppressed by NaCl concentrations. It should be pointed out that collected research data has showed that the codons encoding arginine have a dramatic influence of heterologous protein expression level in E. coli, so it is normally recommended to change the AGG and AGA codon for arginine to CGT in the target gene [23]. Not merely applied in E. coli, similar codon optimization effect was also observed in yeast. Persea americana var. drymifolia defensing (PaDef) was expressed in P. pastoris, whose codon was optimized and the yield of recombinant PaDef reached 79.6 mg/L with antimicrobial activities against both Gram-positive and Gramnegative bacteria [24]. Another codon optimization trick that is always used in E. coli or yeast is adding multiple stop codons to provide strong termination efficiency during the translation process. For instance, TAATGA instead of TAA or TGA alone was normally used in E. coli to avoid the possible low yield of AMP products by the insufficient translational termination [25]. As the development of bioinformatics, codons optimization can be easily performed using online resources, such as https://hpcwebapps.cit.nih. gov/dnaworks/.

2. Expression strategies

2.2. Host strains

2.1. Codon optimization

Besides codon optimization of AMP sequence, using different host strains containing tRNAs for rare codons is another way to solve the low expression problems caused by codon bias. For example, EC-proHeps originally failed for expression in BL21(DE3) cells because it contains three low-usage codons, AGG, CCC, and GGA. This expression issue was later solved by the expression in Rosetta (DE3) cells [26], as Rosetta (DE3) cells contain higher abundance of tRNAs for these three rare codons than those of BL21 (DE3) cells, facilitating the expression of EC-proHeps.

Because of the different abundance of tRNAs in various organisms such as E. coli and yeast, each organism has its own codon preference. Thus, codon optimization is a good strategy to be used to enhance the heterologous expression of target AMPs (Fig. 3A). Previous studies on human beta-defensin-2 (hbd2), human betadefensin-5 (hbd5) and human beta-defensin-6 (hbd6) that were expressed in E. coli have demonstrated that the expression level of

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T. Deng et al. / Protein Expression and Purification 140 (2017) 52e59

Other than different strains of one specie, such as BL21 (DE3) cells and Rosetta (DE3) cells both belonging to the E. coli, different host strains from various species, such as E. coli, Bacillus, and yeast, were also applied in AMPs production. About ten years ago, only about 400 proteins were expressed using P. pastoris [27]. But in recent years, P. pastoris brought more attention in the heterologous expression of antimicrobial peptides, due to its strong secretion and glycosylation properties. A large number of successful cases have shown that better expressions of AMPs were obtained in P. pastoris than those in E. coli. For example, Scygnodin, an AMP segregated from the crayfish, was expressed 1.3 times higher in P. pastoris than that of E. coli [28]. It has to be pointed out that the secretion of the scygnodin to the culture medium simplifies the later purification process. More importantly, it was also indicated that AMP products expressed in P. pastoris presented higher antimicrobial activities compared to that in E. coli, probably due to the post-translational modifications in P. pastoris, such as glycosylation. Research showed that PaDef was expressed abundantly both in E. coli and P. pastoris, but only the products expressed in P. pastoris presented effective antimicrobial activities [24]. Not only better than the E. coli cells, the superiority of P. pastoris over S. cerevisiae was also presented in some other cases. For instance, Ch-penaeidin (CHP), which showed antimicrobial activities against Gram-positive bacteria, Gram-negative bacteria and some fungi, was successfully expressed in both P. pastoris and S. cerevisiae, with higher secreted production of about 100 mg/L in P. pastoris [29]. Another example is that EnterocinL50A and EnterocinL50B could both be expressed in P. pastoris and S. cerevisiae, while the bioactivities of AMPs products obtained from P. pastoris were 6 and 60 times higher, respectively [30,31]. Other than E. coli and yeast, Bacillus is also used for the heterologous expression of AMPs. CecropinB2 (cecB2) could be expressed in various host cells, but it was found that the yield of cecB2 in B. subtilis is higher than that in E. coli, with the yield of 1128 mg/L over 1055 mg/L [32]. Similarly, cecropin A-melittin mutant (CAM-W) was expressed in B. subtilis W700 to reach a yield concentration of 159 mg/L [33]. 2.3. Promoters using in host cells Different promoters are preferred in different host cells (Table 2). The pET series expression vectors were commonly used when expressing AMPs in E. coli cells, as the strong T7 promoter can significantly increase the transcription levels. Scygonadin, an anionic AMP, was constructed in different vectors containing promoters of either T7 or Trc. The yield of Scygonadin was 10.6 mg/L under the Trc promoter, while that was 65.9 mg/L under the T7 promoter, which was 5 times higher [34]. In B. bacillus, P43 is a constitutive strong promoter that was normally used for heterologous protein expression. The use of P43 promoter in expressing Cerceopin AD gave a yield of 30.6 mg/L [35]. Additionally, CAM-W was produced in B. bacillus with a yield of 159 mg/L using the inducible Glv promoter, which could be induced by maltose [33]. Yeast are good host cells due to their strong secretion abilities. In Yeast, two types of promoters are normally used, inducible and constitutive ones. Normally, the inducible promoter is favorably used, as it is convenient to manipulate the procedure of expression, thus enhancing the product yield. When expressing the clavMO in P. pastoris, it was indicated that using the inducible promoter AOX1 could result in a 1.5-fold higher final cell OD compared to the constitutive promoter GAP, thus generating more AMP product [36]. The highest expression level of AMPs utilizing GAP promoter reported so far was expressing the MP1102 in P. pastoris, an AMP designed with three mutations compared to NZ2114, which reached a final yield of 807.42 mg/L [37]. Comparably, the highest

expression level of AMPs utilizing AOX1 promoter was expressing the NZ2114 in P. pastoris, reaching a yield of 2390 mg/L, which was approximate three times than MP1002 [38]. The low cost of methanol leads to the wide application of AOX1 inducible promoter in both laboratory and industrial scales. However, most other inducible promoters, such as T7, Glv, GAL, were not preferred in industrial production, mainly due to the high price of their correspondent inducers. In practical production, constitutive promoters are highly preferred in industrial scale of AMP expression. 2.4. Tandem multimeric expression Tandem multimeric expression is another strategy that is commonly used for high AMPs production. In this strategy, gene copies of the AMPs in the host cells were multiplied to increase the gene transcription level, whereas improved the protein expression yield (Fig. 3B). Research on the expression of antimicrobial peptide CM4 in E. coli BL21(DE3) indicated that the yield of CM4 reached 68 mg/L when the number of gene copy of CM4 was increased to three, which was near four times of the cells containing only one gene copy of CM4 [39]. Similar results were reported that increasing the gene copy number of ABP-dHC-cecropin A to three could enhance its expression yield in E. coli to 300 mg/L in largescale fermentation [40]. In P. pastoris, Plectasin, an AMP possessing the antimicrobial activity against S. suis and S. aureu, was secreted with 8 copies of gene fragments, presenting a yield of 146 mg/L. Comparably, the yeast cells that only contained one gene copy of plectasin gave a yield of 50 mg/L, and the expression of plectasin reached 92 mg/L in E. coli [41]. Not merely happened in E. coli and P. pastoris, high expression caused by tandem multimeric genes was also observed in B. subtilis. For example, Tachyplesin was highly expressed in B. subtilis using tandem multimeric strategy [42]. Although tandem multimeric strategy has been proved to be an efficient method to obtain high expression of AMPs, certain heterologous expression system has to be chosen for specific AMPs. Adenoregulin (ADR), a 33 amino acid antibiotic peptide, was attempted to be expressed both in E. coli BL21(DE3) and P. pastoris GS115 with 2, 4, or 6 tandem repeats. However, none of the monomeric and multimeric ADRs could be expressed in E. coli, but it could be efficiently expressed and secreted in P. pastoris [43]. Another point needs to be emphasized is that no linear correlation exists between the gene copy numbers and the expression levels of target protein. For example, mixed outcomes of the soluble expression of the hBD2 in E. coli were obtained when 1 to 4 gene copies were tested [44]. The studies suggested that pGEX-4T-2 construct with one or two joined hBD2 genes could lead to a higher level expression than cells carrying four joined hBD2 genes. In addition, cells bearing fewer gene copies of hBD2 presented a faster growth rate, indicating that the high expression of AMPs might bring burden to the host cells. Because of these reasons, appropriate gene copy number has to be determined for specific AMPs in a certain heterologous expression system to obtain the highest AMPs production. Although AMPs have been successfully expressed in S. cerevisiae, such as Pediocin PA-1 [45], Plantaricin 423 [46], and b-Defensin-1 [47], tandem multimeric strategy was not effectively applied in S. cerevisiae. The main reason might be related to the high recombination frequency in S. cerevisiae, which could cause the mutual recombination among the tandem multimeric genes. 2.5. Fusion tag expression Two other challenging questions of the heterologous expression of AMPs are the toxicity of the AMPs to the host cells and the

T. Deng et al. / Protein Expression and Purification 140 (2017) 52e59

susceptibility to degradation due to their low molecular weight and high cationic properties [48]. In order to solve these two obstacles, an effective strategy of expressing AMPs with fusion tags was developed (Fig. 3C). Utilization of fusion tags will normally not affect the activity of antimicrobial peptides as these fusion tags can be easily removed by chemical agents or specific proteases recognizing its correspondent cleavage sites [49,50]. The normally used fusion tags include maltose-binding protein (MBP), glutathione Stransferase (GST), elastin like polypeptide (ELP), small ubiquitin like modifier (SUMO), thioredoxin A (TrxA) (Table 3), which can facilitate not only the folding of target AMPs, but also the later purification process. Comparing to AMP itself that normally contains less than 30 amino acids, purification of fusion tag fused AMPs is much easier. In fact, commercial available amylose resin and glutathione resin have been developed to further simplify the purification procedure of MBP and GST fused AMPs, respectively. Many successful cases have been published for using the fusion protein to help the expression of AMPs. As a suitable chaperone for cationic peptide expression, TrxA can outset high net positive charge distributed on AMPs surface, which is beneficial to highefficient express. Under optimized expression conditions in E. coli, a high percentage (>60%) of soluble TrxA-omHD6 fusion protein was obtained with a yield close to 169 mg/L [51]. Another case is that the recombinant buforin IIb using TrxA as the fusion tag could achieve about 3.1 mg/L in E. coli [52]. Fusion tag strategy is also proved to be efficient in B. subtilis. For example, using SUMO as a fusion tag, which could be cleaved by SUMO protease, the expression of Cathelicidin-BF (CBF) achieved 3 mg/L in culture medium, and presented antimicrobial activity against E. coli ATCC25922, E. coli K 88, E. coli K12, Pseudomonas aeruginosa CMCC27853, Staphylococcus aureus ATCC25923, and Staphylococcus epidermidis ATCC12228 [53]. Similarly, antimicrobial peptide NZ17074 was expressed in P. pastoris after being fused with SUMO3, reaching a yield of 4.1 mg/L [54]. An interesting phenomenon is that AMPs fusing to different fusion tags might generate different outcomes. ADR fused to TrxA fusion tag was expressed successfully in E. coli BL21 (DE3) but could not be expressed in the same host cells when fused to the GST fusion tag [43]. More interestingly, it has been recently reported that fusion protein might enhance the antimicrobial activity of AMPs. The antibacterial activity of TPI against B. subtilis (CMCC63501) was enhanced after fused with human lysozyme to form human lysozyme-tachyplesin I fusion protein [55]. All these studies indicated that the characteristics of fusion tags and interaction between target protein and fusion tags should be considered when choosing the right fusion tag for AMP production.

57

2.6. Hybridization expression Recently, a novel strategy, hybridization expression, was developed to enhance the expression level and introduce new properties of AMPs. In the concept of hybridization expression, two antimicrobial peptides with different properties are fused to generate a new AMP that present not only high expression yield but with increased antimicrobial activity and low cytotoxicity against host cells (Fig. 3D). Cecropins, an AMP family comprised of 31e39 amino acids that present antimicrobial activities against both Gram-negative and Gram-positive bacteria is widely used as part of hybrid AMPs [56]. For example, segments of cecropin A combining with segments of melittin could generate antibacterial, antifungal and potential anti-tumor activities, expanding their antimicrobial spectra against E. coli, X. vesicatoria, and P. syringee. Besides, a novel hybrid AMP, LF15-CA8, which consists of the 1e8 amino acid residues of cecropin A and the 1e15 amino acid residues of LfcinB, could reach a yield of 10 mg/L after purification in E. coli, and demonstrated an increased antibacterial activity against Grampositive bacterium (Staphylococcus aureus ATCC25923) without showing obvious hemolytic activity against human erythrocytes [57]. In P. pastoris, hybrid peptide cecropin A (1e8)-magainin2 (1e12) (CA-MA) was expressed and obtained 22 mg/L active pure CA-MA ultimately [58]. CA-MA displayed antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. In B. subtilis, cecropin AD that was composed of cecropinA and cecropinD was expressed at a yield of 30.6 mg/L, demonstrating antimicrobial activity toward Staphylococcus aureus and E. coli [35]. These successful examples indicated the promising future for AMP engineering.

3. Summary and outlook The abuse of traditional antibiotics in medicine, food and aquaculture has raised various problems, ranging from health to environment. Due to their broad antimicrobial spectrum and specific antimicrobial mechanism, microbes are not easy to develop resistance against AMPs, which leads AMPs to be preferred replacements for the traditional antibiotics. To expand the application of AMPs, major problems that need to be solved include low yield and purity, and high production cost during the traditional extraction method. As a good solution to these problems, many expression strategies for heterologous expression of AMPs have been developed nowadays, among which the codon optimization, different host cells, optimized promoters, multimeric gene copies, fusion proteins, and AMP hybridization are studied. Although good progress has been achieved, different strategies against specific

Table 3 Characteristics of recombinant protein fusion tags for AMPs' production. Tag

Tag placement

AMP

Host cell

Yield(mg/L)

Advantages

Ref.

MBP

N- or C- terminus N- or C- terminus

ELP

N- or C- terminus

SUMO

N- or C- terminus

TrxA

N- or C- terminus

BL21 (DE3) BL21(DE3) BL21 (DE3) BL21 (DE3) BL21 (DE3) BL21 (DE3) BL21 (DE3) BL21 (DE3) BL21 (DE3) BL21(DE3) BL21 (DE3) BL21(DE3) BL21(DE3)

0.9 3.0 ND 1.69 117 0.6 1.8 12 287.9 23 1.0 8.0 ND

Enhanced solubility of target, do not leave extraneous amino acids

GST

Crotamine ORBK palustrin-2CE DS4 Pa-MAP 2 moricin CM4 hbd4 cecropin AD ABP-dHC-cecA M-L Ranalexin Ib-AMP4 snakin-2

[84] [85] [86] [87] [88] [89] [89] [90] [40] [91] [92] [93] [94]

ND: not determined.

Enhanced solubility of target, highly specific binding amino acids Cost effective than conventional purification

Enhanced target solubility, unique tag removal method Helps in crystallization, Aid in refolding of target protein

58

T. Deng et al. / Protein Expression and Purification 140 (2017) 52e59

AMPs have to be established according to the physical and chemical properties of AMPs. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31540068 to L.Y.) and Natural Science Foundation of Hubei Province of China (No. 2015CFA088 to L.Y.). Conflict of interest The authors declare that there are no conflicts of interest.

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