Protein Expression and Purification 87 (2013) 72–78

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Protein Expression and Purification

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Production of human antimicrobial peptide LL-37 in Escherichia coliusing a thioredoxin–SUMO dual fusion system

Yifeng Li ⇑Protein Production Core Facility, Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States

a r t i c l e i n f o

Article history:Received 5 September 2012and in revised form 22 October 2012Available online 7 November 2012

Keywords:Antimicrobial peptideDual-tagFusion proteinLL-37SUMOThioredoxin

1046-5928/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.pep.2012.10.008

⇑ Address: Medical Building 431B, Department ofTexas Health Science Center at San Antonio, 7703 Floy78229, United States.

E-mail addresses: yifgli@gmail.com, liy10@uthscsa1 Abbreviations used: hCAP18, human cationic antimi

glutathione transferase; Trx, thioredoxin; SUMO, smaIPTG, isopropyl-b-D-thiogalactopyranoside; SDS–PAGEacrylamide gel electrophoresis; MIC, minimum inhibit

a b s t r a c t

LL-37 is a human antimicrobial peptide that has been shown to possess multiple functions in hostdefense. In this report, the peptide was expressed as a fusion with a thioredoxin–SUMO dual-tag. UponSUMO protease mediated cleavage at the SUMO/peptide junction, LL-37 with its native N-terminus wasgenerated. The released peptide was separated from the dual-tag and cleavage enzyme by size-exclusionchromatography. Mass spectrometry analysis proves that the recombinant peptide has a molecularweight as theoretically expected for its native form. The produced peptide displayed antimicrobialactivity against Escherichia coli K-12. On average, 2.4 mg peptide was obtained from one liter of bacterialculture. Thus, the described approach provides an effective alternative for producing active recombinantLL-37 with its natural amino acid sequence in E. coli.

� 2012 Elsevier Inc. All rights reserved.

Introduction

LL-37 is a human antimicrobial peptide primarily produced byphagocytic leukocytes and epithelial cells. The peptide is initiallysynthesized as a precursor named human cationic antimicrobialprotein of 18 kDa (hCAP18)1 and the active form, which resides inthe C-terminus, is released by proteolytic cleavage [1]. In additionto antimicrobial activity, LL-37 demonstrates diverse immunomodu-latory properties such as the ability to mediate chemotaxis, acceler-ate angiogenesis and promote wound healing [2,3]. As an essentialmultifunctional molecule, LL-37 has attracted great interest for thetherapeutic and diagnostic value it has [4–9].

Many antimicrobial peptides have been obtained through re-combinant production in various heterologous hosts, among whichEscherichia coli has been the most widely used one [10–13]. Due totheir degradation-prone and bactericidal properties, antimicrobialpeptides produced in E. coli are often expressed as fusion proteins[11–13]. Bacterial production of LL-37 using various fusion part-ners including glutathione transferase (GST) [14,15], thioredoxin(Trx) [16–21], family III cellulose-binding module (CBM) [22] andsmall ubiquitin-related modifier (SUMO) [23,24] was previously

ll rights reserved.

Biochemistry, University ofd Curl Drive, San Antonio, TX

.educrobial protein of 18 kDa; GST,ll ubiquitin-related modifier;

, sodium dodecylsulfate–poly-ory concentration.

reported. Among the above-mentioned carriers that are commonlyused for fusion expression of antimicrobial peptides, SUMO is a rel-atively new one [12]. As a fusion partner, SUMO was found to im-prove the folding and solubility of the target protein [25,26]. Inaddition, the existence of SUMO protease offers a unique advan-tage to this system. Unlike other commonly used proteases, whichrecognize short sequence motifs, SUMO protease only recognizesSUMO’s tertiary structure and cleaves right after the Gly-Gly se-quence at the C-terminus of SUMO. As a result, the enzyme nevercleaves within the protein of interest and allows the production oftarget protein with native N-terminus when the fusion is properlydesigned [25]. The application of SUMO fusion system to antimi-crobial peptides production in E. coli was recently described in apatent application [23] and a research article [24] by a group atEmory University. However, whereas LL-37 was one among severalantimicrobial peptides used to demonstrate the feasibility of thissystem, detailed information about fusion protein cleavage and fi-nal peptide yield was not provided in either document.

In order to confirm the feasibility and efficiency of SUMO fusionsystem for LL-37 production, I made a construct for SUMO-LL37expression (Fig. 1A). It was found that the protein expression levelwas significantly lower than that of the Trx-LL37 fusion [21] and aportion of the expressed fusion protein degraded into smaller frag-ments during purification (data not shown). A new construct forTrx-SUMO-LL37 expression (Fig. 1B) was subsequently made tosolve these problems. This Trx and SUMO dual-tagged LL-37 fusionwas overexpressed, stable and efficiently cleaved upon treatmentwith SUMO protease. The released peptide can be separated fromthe dual-tag and SUMO protease (in the form of MBP fusion) by

B

A

Fig. 1. Schematic representation of (A) SUMO-LL37 and (B) Trx-SUMO-LL37 fusionproteins. In both designs, SUMO protease will cleave after the Gly-Gly sequence atthe C-terminus of SUMO, allowing LL-37 with its native N-terminus to be releasedfrom the corresponding fusion protein.

Y. Li / Protein Expression and Purification 87 (2013) 72–78 73

size-exclusion chromatography. The produced peptide displayedantimicrobial activity against E. coli K-12. On average, 2.4 mg pep-tide was obtained from one liter of bacterial culture. Thus, the de-scribed approach provides an effective means for producing activerecombinant LL-37 with its natural amino acid sequence.

Materials and methods

Materials

Rosetta competent cells were obtained from EMD Millipore.JM109 competent cells were purchased from Promega. E. coli K-12 was obtained from American Type Culture Collection (ATCC).

A

B

C

Fig. 2. Expression/cloning region of (A) pET-28a and (B) its modified version, in which a sBamHI sites. This modification allows the vector to be used for SUMO fusion expressiocoding sequence was digested with BsaI and BamHI, and ligated into the modified pET-2

Plasmids pET-28a and pET-32a were purchased from Novagen.The plasmids pJOE4905.1 (which encodes MBP and SUMO dual-tagged GFP) and pJOE4847.2 (which encodes MBP tagged SUMOprotease Ulp1) were kind gifts of Dr. Altenbuchner (UniversitätStuttgart, Germany). Oligonucleotide primers were synthesized atthe Nucleic Acids Core Facility at the University of Texas HealthScience Center at San Antonio (UTHSCSA). Vent DNA polymerase,restriction enzymes, calf intestinal alkaline phosphatase (CIP),quick ligation kit, dNTP mix and DNA marker were obtained fromNew England Biolabs. QIAquick PCR purification kit, QIAquick gelextraction kit, QIAprep spin miniprep kit, and Ni–NTA agarosewere purchased from Qiagen. Difco LB broth was purchased fromBD Biosciences. Ampicillin sodium salt was purchased fromAffymetrix. Tris, glycine, SDS, 30% acrylamide/bis, and proteinstandards were purchased from Bio-Rad. Isopropyl b-D-1-thioga-lactopyranoside (IPTG) and L-rhamnose monohydrate were pur-chased from Gold Biotechnology and Sigma–Aldrich, respectively.

Expression vector construction

In order to generate the construct for SUMO-LL37 fusionexpression (Fig. 1A), the commercial vector pET-28a was firstmodified by inserting a sequence encoding Smt3, the yeast SUMOprotein (Fig. 2A and B). This was achieved by amplifying the Smt3coding sequence with primers 1 and 2 (Table 1) using plasmidpJOE4905.1 as the template and inserting the NdeI and BamHI

equence encoding yeast SUMO protein Smt3 was inserted between the NdeI and then. (C) Illustration of SUMO-LL37/pET-28a vector construction. PCR amplified LL-37

8a plasmid doubly digested with the same enzymes.

Table 1Primers used for gene amplification.

Primers Sequences (50–30)

1 GGAATTCCATATGGAGGTCAAGCCAGAAGTCAAGCCTNdeI

2 CGCGGATCCCCACTAGTGGTCTCAGCCACCAATCTGTTCACGATGGGCCTCAATBamHI BsaI

3 GGTCTCCCTGGCCTGCTGGGTGATTTCTTCCGGAAATCTBsaI

4 CGCGGATCCTCAGGACTCTGTCCTGGGTACAAGATTCCGBamHI

5 GTGCTGGCCATCATCATCATCATCATCATCACAGCAGCMscI

Note: The restriction sites used for cloning are shown in italics, with the name of the enzyme under the sequence. Inprimer 2, the created BsaI site is underlined.

A

B

Fig. 3. Expression/cloning region of (A) pET-32a and (B) the construct for Trx-SUMO-LL37. The DNA sequence encoding SUMO-LL37 was inserted into pET32a between theMscI and BamHI sites.

74 Y. Li / Protein Expression and Purification 87 (2013) 72–78

doubly digested PCR product into similarly digested pET-28a. ABsaI restriction site was created at the end of the codingsequence for SUMO (Fig. 2B). Next, the LL-37 coding sequencewas PCR amplified with primers 3 and 4 (Table 1) using a vectorharboring the cDNA for full-length hCAP18 (GenBank accessionnumber Z38026) as template, and the PCR product was digestedwith BsaI and BamHI and ligated into the modified pET-28aplasmid doubly digested with the same enzymes (Fig. 2C). Theresultant construct for SUMO fusion expression was namedSUMO-LL37/pET-28a.

For fusion expression of LL-37 with Trx and SUMO dual-tag(Fig. 1B), the gene encoding SUMO-LL37 was amplified by PCR withprimers 5 and 4 (Table 1) using SUMO-LL37/pET-28a as template.

The PCR product was digested with MscI and BamHI, and ligatedinto pET-32a doubly digested with the same enzymes (Fig. 3).The resultant construct was named SUMO-LL37/pET-32a.

The presence and identity of the insert in both constructswas verified by diagnostic restriction digestion and DNAsequencing.

Fusion protein expression and purification

Rosetta competent cells harboring the expression vector SUMO-LL37/pET-32a were grown overnight in 100 ml LB broth (supple-mented with 100 lg/ml ampicillin) at 37 �C. This overnight culturewas used to inoculate one liter of fresh LB medium and cells were

1 2 3

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Fig. 4. (A) Relative expression levels of SUMO-LL37 and Trx-SUMO-LL37 revealed by SDS–PAGE analysis. Lane 1 and 2, lysate of cells harboring SUMO-LL37/pET-28a andSUMO-LL37/pET-32a (protein expression was induced under identical conditions), respectively; Lane 3, protein standards. (B) Expression and purification of Trx-SUMO-LL37fusion protein as followed by tricine SDS–PAGE (12.5%). Lane 1, whole cell lysate; Lane 2, cell lysate supernatant; Lane 3, imidazole eluate; Lane 4, size-exclusionchromatography purified fusion protein; Lane 5, protein standards. (C) Size-exclusion chromatography elution profile of Trx-SUMO-LL37 fusion (HiLoad 16/60 Superdex 75column). Inset shows SDS–PAGE analysis of fractions that cover the major peak. The two minor peaks represent low molecular weight contaminants and salts, respectively.

Y. Li / Protein Expression and Purification 87 (2013) 72–78 75

grown at 37 �C with shaking at 250 rpm. Target protein expressionwas induced by adding IPTG to a final concentration of 1 mM whenthe culture OD600 (optical density at 600 nm) reached 0.6. Afteradditional 5 h cultivation at 37 �C, cells were harvested by centri-fugation at 5000 rpm for 10 min. The bacterial pellet (�4.8 g)was resuspended in 30 ml of lysis buffer (25 mM Tris, 200 mMNaCl, pH 8.0) and cells were disrupted by sonication. Triton X-100 was added to a final concentration of 1% at this point. The celllysate was then shaken at 4 �C for 30 min followed by centrifuga-tion at 20,000 rpm for 40 min. The supernatant was combined with5 ml Ni–NTA resin suspension and shaken at 4 �C for 2 h. Afterbeing spun down, the resin was washed twice with 35 ml of lysisbuffer containing 40 mM imidazole by shaking at 4 �C for 30 min.The fusion protein was eluted with 20 ml of lysis buffer containing500 mM imidazole. The eluate was run through a HiLoad 16/60

Superdex 75 column equilibrated with cell lysis buffer, whichremoves contaminants left over from the initial affinitypurification.

Expression and purification of SUMO proteases

MBP-Ulp1 (SUMO protease in the form of MBP fusion) was ex-pressed and purified following the protocol developed by Motej-added and Altenbuchner [27]. In brief, E. coli strain JM109harboring recombinant vector pJOE4847.2 was grown at 37 �C un-til OD600 reached 0.4, at which point 0.2% (w/v) L-rhamnose wasadded and the culture temperature was reduced to 30 �C. After18 h, bacteria were harvested and resuspended in cell lysis buffer.Cells were lysed by sonication and the cell lysate supernatant wasincubated with Ni–NTA resin for 3 h with shaking at 4 �C. After

76 Y. Li / Protein Expression and Purification 87 (2013) 72–78

washing twice with 25 mM imidazole, the protein bound to the re-sin was eluted with cell lysis buffer containing 300 mM imidazole.

Peptide release, isolation and characterization

Trx-SUMO-LL37 fusion protein was cleaved with MBP-Ulp1 at amass ratio of 10:1 (i.e., for 1 mg of purified fusion, 0.1 mg proteasewas added). The cleavage reaction was allowed to proceed at roomtemperature for 18 h. After cleavage, the sample volume was re-duced to half of its original value using a centrifugal filter deviceand the concentrated cleavage mixture was subjected to a Super-dex 200 10/300 GL gel filtration column for peptide isolation(1 ml of sample was loaded each time). The eluted fractions con-taining the target peptide from different runs were combinedand lyophilized for further use. The identity of the purified recom-binant LL-37 was checked by mass spectrometry analysis.

Antibacterial activity assay

Antimicrobial activity of the recombinant LL-37 was evaluatedby determining the minimal inhibitory concentration (MIC) as pre-viously described [17,21]. In brief, E. coli K-12 in log phase was di-luted with LB media and partitioned into a 96-well plate (90 ll perwell). Dilution and partition of the bacterial culture was made insuch a way that each well contains approximately 105 cells (it is as-sumed that OD600 of 1.0 equals �109 cells per ml). A LL-37 stocksolution was made by solubilizing the lyophilized peptide in phos-phate buffered saline (PBS). Peptide at five designed concentrationswas prepared by serial dilution of the stock solution. To each wellof bacterial culture, 10 ll of the peptide solution was added. Ateach peptide concentration, the assay was repeated three times.The plate was read at 620 nm after incubation at 37 �C overnight.

Results

Fusion protein expression and purification

The Trx and SUMO dual-tagged LL-37 (encoded by SUMO-LL37/pET-32a) was overexpressed (Fig. 4A) and can be extracted to thesoluble portion by adding 1% Triton-X 100 (detergent is criticalfor solubilization of the fusion because of LL-37’s tendency formembrane association) to the cell lysate (Fig. 4B, lane 2). Aftersequential affinity and size exclusion chromatography steps, the

LL-37

uncut fusion

Trx-SUMO tag

1 2

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MBP-Ulp1MBP-Ulp1

Fig. 5. Recombinant SUMO protease and its mediated cleavage of the Trx-SUMO-LL37 fuquality checked by glycine SDS–PAGE (12%). (B) Cleavage efficiency of Trx-SUMO-LL37 fPAGE (12.5%). The reactions were allowed to proceed for 18 h at room temperature. Laprotein, respectively; Lane 6, protein standards.

fusion protein was obtained in high purity (Fig. 4B, lane 4). The ma-jor peak in the gel filtration elution profile represents the fusionprotein (Fig. 4C).

Peptide release, isolation and characterization

The SUMO protease as an MBP fusion has proved to be enzy-matically active [27]. Following the protocol developed by Motej-added and Altenbuchner [27], MBP-Ulp1 was obtained inrelatively pure form after a single step affinity purification usingNi–NTA resin (Fig. 5A). In a small-scale test, it was learned thatthe cleavage reaction reached completion in 18 h at room temper-ature when approximately 0.1 mg MBP-Ulp1 per mg Trx-SUMO-LL37 fusion protein was used (Fig. 5B).

Taking advantage of LL-37’s strong aggregation tendency, thereleased peptide was separated from the dual-tag and MBP-Ulp1using the Superdex 200 10/300 GL gel filtration column (Fig. 6A).The results from large-scale cleavage and final peptide purificationwere summarized in Fig. 6B. MALDI–TOF MS analysis (see supple-mentary data) proved that the peptide has a measured mass(4493.55) that agrees well with its calculated theoretical value(4493.28). The average yields for purified fusion protein and LL-37 from one liter of culture was 37.8 and 2.4 mg, respectively(see Table 2).

Antibacterial activity assay

The recombinant LL-37 produced in this study contains a se-quence identical to its natural form (no extra residue was left overat its N-terminus from the SUMO protease cleavage). In all repli-cates, the MIC value of this peptide against E. coli K-12 was foundto be 40 lM, which is identical to the corresponding value obtainedusing a synthetic peptide with the native sequence and a recombi-nant peptide with an extra Pro residue at its N-terminus [17].

Discussion

Production of antimicrobial peptides in E. coli is challenging.These peptides’ bactericidal nature makes them potentially fatalto the producing host; and their small size and high cationic prop-erty makes them highly susceptible to proteolytic degradation. Astrategy that effectively overcomes both obstacles is to fuse thepeptide of interest to a carrier protein [11–13]. The target peptide

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sion protein. (A) Purified recombinant SUMO protease (in the form of MBP fusion)usion protein with different amounts of SUMO protease as followed by tricine SDS–ne 1–5, 0, 30, 60, 90, 120 lg of recombinant protease was added to 1 mg of fusion

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Fig. 6. (A) Size-exclusion chromatography elution profile of SUMO protease cleavage mixture (Superdex 200 10/300 GL column). Inset shows SDS–PAGE analysis of peak 1, 2and 3. The first peak contains the target peptide. The second peak contains SUMO protease and some of the carrier protein, and the third peak contains the major portion ofTrx-SUMO fusion carrier. (B) Fusion cleavage and peptide purification as followed by tricine SDS–PAGE (12.5%). Lane 1, affinity and size-exclusion chromatography purifiedfusion protein; Lane 2, SUMO protease cleavage mixture; Lane 3, purified LL-37; Lane 4, protein standards.

Table 2Average yields of fusion protein and final purified LL-37 from 1L of culture.

Purification step Trx-SUMO-LL37 (mg) LL-37 (mg) Purity (%)

After fusion purification 37.8 NAa >95After peptide purification NA 2.4 >95

a Not applicable.

Y. Li / Protein Expression and Purification 87 (2013) 72–78 77

can be released from the fusion at a later stage by enzymatic orchemical cleavage at corresponding site around the carrier-peptidejunction. Due to the aggregation tendency of many antimicrobial

peptides, enzymatic cleavage was generally found less efficientthan chemical means [28]. In consistent with this observation,LL-37 expressed as GST and Trx fusions were cleaved with low effi-ciency by factor Xa and enterokinase, respectively [15,19].Although chemical cleavage is usually efficient, it is less specificand can cause side-chain modification. Both enzymatic and chem-ical cleavages may leave one or two non-native residues at the N-terminus of the target peptide.

Recently, SUMO has been used as a novel fusion carrier for theproduction of recombinant proteins as it can promote solubleexpression of the target [25,26]. In addition, SUMO protease ishighly specific and efficient at removing the carrier, and generates

78 Y. Li / Protein Expression and Purification 87 (2013) 72–78

target protein with native N-terminus. Furthermore, SUMO prote-ase can be produced in large quantities with relative ease, which isan advantage for large-scale protein production using this system.The application of SUMO fusion system to antimicrobial peptidesproduction in E. coli was previously reported in a patent applica-tion [23] and a research article [24]. However, whereas LL-37was one of the antimicrobial peptides used for demonstration, nodetailed information about fusion protein cleavage and final pep-tide yield was provided. Therefore, the feasibility of SUMO fusionsystem for LL-37 production has not been convincinglydemonstrated.

In hopes of getting first-hand information about LL-37 produc-tion using SUMO system, I made the fusion construct as shown inFig. 1A. It was learned that the expression level of SUMO-LL37 wassignificantly lower than that of the Trx-LL37 fusion [21] and a por-tion of the expressed fusion protein degraded into smaller frag-ments during purification (data not shown). A new construct forTrx-SUMO-LL37 expression (Fig. 1B) was subsequently made tocircumvent these problems. This Trx and SUMO dual-tagged LL-37 fusion was overexpressed (Fig. 4A and B), stable and efficientlycleaved by SUMO protease (Fig. 5B). The released peptide can beseparated from the dual-tag and MBP-Ulp1 by size-exclusion chro-matography. LL-37 produced using the described approach, whichhas amino acid sequence identical to its native form, displayedantimicrobial activity against E. coli K-12. This novelly-designedTrx and SUMO dual fusion system has certain advantages. Thepresence of SUMO tag allows the target peptide to be released bySUMO protease mediated cleavage whereas adding the Trx taggreatly improves fusion protein expression and stability. On aver-age, 2.4 mg highly purified peptide was obtained from one literof bacterial culture. Thus, this new approach provides an effectivealternative for producing active recombinant LL-37 with its naturalamino acid sequence in E. coli.

Acknowledgments

This work was supported by departmental funding dedicated tothe protein production core facility. The author is grateful to Dr.Ole Sørensen (Lund University, Lund, Sweden) for the cDNA ofhCAP18 and Dr. Altenbuchner (Universität Stuttgart, Germany)for plasmids pJOE4905.1 (which encodes MBP and SUMO dual-tagged GFP) and pJOE4847.2 (which encodes MBP tagged SUMOprotease Ulp1). The author would like to thank Sammy Pardoforfor mass spectrometry analysis.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.pep.2012.10.008.

References

[1] O.E. Sørensen, P. Follin, A.H. Johnsen, J. Calafat, G.S. Tjabringa, P.S. Hiemstra, N.Borregaard, Human cathelicidin, hCAP-18, is processed to the antimicrobialpeptide LL-37 by extracellular cleavage with proteinase 3, Blood 97 (2001)3951–3959.

[2] U.H. Dürr, U.S. Sudheendra, A. Ramamoorthy, LL-37, the only human memberof the cathelicidin family of antimicrobial peptides, Biochim. Biophys. Acta1758 (2006) 1408–1425.

[3] R. Bucki, K. Leszczynska, A. Namiot, W. Sokołowski, Cathelicidin LL-37: amultitask antimicrobial peptide, Arch. Immunol. Ther. Exp. (Warsz) 58 (2011)15–25.

[4] A. Nijnik, R.E. Hancock, The roles of cathelicidin LL-37 in immune defences andnovel clinical applications, Curr. Opin. Hematol. 16 (2009) 41–47.

[5] P. Méndez-Samperio, The human cathelicidin hCAP18/LL-37: a multifunctionalpeptide involved in mycobacterial infections, Peptides 31 (2010) 1791–1798.

[6] W.K. Wu, G. Wang, S.B. Coffelt, A.M. Betancourt, C.W. Lee, D. Fan, K. Wu, J. Yu,J.J. Sung, C.H. Cho, Emerging roles of the host defense peptide LL-37 in humancancer and its potential therapeutic applications, Int. J. Cancer 127 (2010)1741–1747.

[7] J.A. Hensel, D. Chanda, S. Kumar, A. Sawant, W.E. Grizzle, G.P. Siegal, S.Ponnazhagan, LL-37 as a therapeutic target for late stage prostate cancer,Prostate 71 (2011) 659–670.

[8] T. Leung, K. Ching, A. Kong, G. Wong, J. Chan, K. Hon, Circulating LL-37 is abiomarker for eczema severity in children, J. Eur. Acad. Dermatol. Venereol. 26(2012) 518–522.

[9] M. Reinholz, T. Ruzicka, J. Schauber, Cathelicidin LL-37: an antimicrobialpeptide with a role in inflammatory skin disease, Ann. Dermatol. 24 (2012)126–135.

[10] A.B. Ingham, R.J. Moore, Recombinant production of antimicrobial peptides inheterologous microbial systems, Biotechnol. Appl. Biochem. 47 (2007) 1–9.

[11] Y. Li, Z. Chen, RAPD: a database of recombinantly-produced antimicrobialpeptides, FEMS Microbiol. Lett. 289 (2008) 126–129.

[12] Y. Li, Carrier proteins for fusion expression of antimicrobial peptides inEscherichia coli, Biotechnol. Appl. Biochem. 54 (2009) 1–9.

[13] Y. Li, Recombinant production of antimicrobial peptides in Escherichia coli: areview, Protein Expr. Purif. 80 (2011) 260–267.

[14] Y. Feng, Y. Yang, N. Huang, Q. Wu, B. Wang, E. coli-based production ofrecombinant FALL-39, Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 20 (2003)634–637.

[15] J.Y. Moon, K.A. Henzler-Wildman, A. Ramamoorthy, Expression andpurification of a recombinant LL-37 from Escherichia coli, Biochim. Biophys.Acta 1758 (2006) 1351–1358.

[16] Y.H. Yang, G.G. Zheng, G. Li, X.J. Zhang, Z.Y. Cao, Q. Rao, K.F. Wu, Expression ofbioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL-37, in Escherichia coli, Protein Expr. Purif. 37 (2004) 229–235.

[17] Y. Li, X. Li, G. Wang, Cloning, expression, isotope labeling, and purification ofhuman antimicrobial peptide LL-37 in Escherichia coli for NMR studies, ProteinExpr. Purif. 47 (2006) 498–505.

[18] Y. Li, X. Li, H. Li, O. Lockridge, G. Wang, A novel method for purifyingrecombinant human host defense cathelicidin LL-37 by utilizing its inherentproperty of aggregation, Protein Expr. Purif. 54 (2007) 157–165.

[19] Y. Li, Expression systems and database for biological, structural andbioinformatic studies of antimicrobial peptides. Ph.D. Dissertation.University of Nebraska Medical Center, Omaha, NE. (UMI No. 3314590), 2008.

[20] J. Krahulec, M. Hyršová, S. Pepeliaev, J. Jílková, Z. Cerny, J. Machálková, Highlevel expression and purification of antimicrobial human cathelicidin LL-37 inEscherichia coli, Appl. Microbiol. Biotechnol. 88 (2010) 167–175.

[21] Y. Li, A novel protocol for the production of recombinant LL-37 expressed as athioredoxin fusion protein, Protein Expr. Purif. 81 (2012) 201–210.

[22] R. Ramos, L. Domingues, M. Gama, Escherichia coli expression and purificationof LL37 fused to a family III carbohydrate-binding module from Clostridiumthermocellum, Protein Expr. Purif. 71 (2010) 1–7.

[23] B. Bommarius, M. Sherman, D. Kalman, X. Cheng, Production of Anti-microbialPeptides, World Pat. WO/2008/140582, 2008.

[24] B. Bommarius, H. Jenssen, M. Elliott, J. Kindrachuk, M. Pasupuleti, H. Gieren,K.E. Jaeger, R.E. Hancock, D. Kalman, Cost-effective expression and purificationof antimicrobial and host defense peptides in Escherichia coli, Peptides 31(2010) 1957–1965.

[25] M.P. Malakhov, M.R. Mattern, O.A. Malakhova, M. Drinker, S.D. Weeks, T.R.Butt, SUMO fusions and SUMO-specific protease for efficient expression andpurification of proteins, J. Struct. Funct. Genom. 5 (2004) 75–86.

[26] T.R. Butt, S.C. Edavettal, J.P. Hall, M.R. Mattern, SUMO fusion technology fordifficult-to-express proteins, Protein Expr. Purif. 43 (2005) 1–9.

[27] H. Motejadded, J. Altenbuchner, Construction of a dual-tag system for geneexpression, protein affinity purification and fusion protein processing,Biotechnol. Lett. 31 (2009) 543–549.

[28] K.L. Piers, M.H. Brown, R.E. Hancock, Recombinant DNA procedures forproducing small antimicrobial cationic peptides in bacteria, Gene 134 (1993)7–13.