Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli

doi:10.1016/j.pep.2005.07.002

Protein Expression and Purification

Volume 45, Issue 2, February 2006, Pages 269-274

https://doi.org/10.1016/j.pep.2005.07.002 Get rights and content

Abstract

Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-terminal homoserine lactone (DCD-1Hsl). Dermcidin coding sequence was cloned downstream of a 125 amino acid ketosteroid isomerase gene and upstream of a His₆Tag sequence in pET-31b(+) vector and transformed into Escherichia coli. The fusion protein was expressed in the form of inclusion bodies, purified on His Bind Resin, and cleaved by CNBr to release recombinant DCD-1Hsl. Purification of rDCD-1Hsl was achieved by solid-phase extraction that yielded milligram amounts of peptide with more than 95% purity. Recombinant peptide showed antimicrobial activities against E. coli ML-35p, Salmonella typhimurium 5156, Listeria monocytogenes 264, S. aureus 29/58 (clinical isolate), and C. albicans K2 (clinical strain). The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of dermcidin in its biologically active form.

Section snippets

Strains and plasmids

Escherichia coli BLR(DE3)pLysS (Novagen) was used as an expression host. E. coli TG1 (Boehringer–Mannheim) was used as a host for subcloning and plasmid amplification. E. coli ML-35p clinical strain (a kind gift from Professor R.I. Lehrer, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, USA); food isolates of Salmonella typhimurium 5156 and L. monocytogenes 264, S. aureus 29/58 (clinical isolate) (obtained from the Department of General

Results and discussion

To establish the E. coli expression system for production of dermcidin, we exploited pET31b+ vector [6]. Sequence coding for a dermcidin variant with N-terminal leucine and C-terminal glutamine (LL-DCD-1Q) was subcloned into AlwNI site downstream of the KSI fusion partner and upstream of the His₆Tag. Introduction of artificial fusion protein or tag sequences, such as KSI fusion partner (enhancing the formation of inclusion bodies) and hexa-histidine (facilitating purification of the fusion

Conclusions

In summary, this work presents the expression and purification method that can be applied for the production of human anionic antimicrobial peptide–dermcidin in bacteria. The above results showed that KSI – DCD-1–His₆Tag protein expression system was able to circumvent the toxicity of DCD-1Hsl on the host cells and simplify the peptide purification procedure. Since isolated DCD-1Hsl exhibited strong antimicrobial activity against tested microorganisms, we can conclude that this

Acknowledgments

We thank Professor Robert Lehrer for providing E. coli ML-35p strain. We are grateful to Dr. Helena Bujdáková for kindly giving the C. albicans K2 clinical strain, and to the colleagues from the Department of General Microbiology of the Institute of Molecular Biology for supplying the food isolates of L. monocytogenes, S. typhimurium, and a kind gift of clinical S. aureus strain.

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There are more references available in the full text version of this article.

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In all cases, P < .05 was considered statistically significant. To establish the E. coli expression system for production of GLP-2, we exploited pET31b + vector is driven by the constitutive hup promoter(Čipáková et al., 2006). To determine the correct cloning of the expression vector, the plasmid was extracted from B. longum transfected with pET31b+/GLP-2 and digested with Nde I and Xho I. However, to ensure the consistency of the plasmid, we linked a replication of Bifidobacterium-pMB1(Rossi et al., 1997) into pET31b+.
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One efficient fusion is ketosteroid isomerase (KSI) [33], a hydrophobic protein that induces the formation of inclusion bodies. This fusion was established with a small peptide mating factor from yeast [34], and efficiently adapted for a hydrogel-forming peptide [35,36], antimicrobial partial sequences from bovine milk lactophoricin [37], antimicrobial human dermcidin [38], an aggregating amyloid protein [39], a highly-charged antimicrobial peptide with sequence PFWRIRIRR [40] as well as an intrinsically disordered domain of human cardiac troponin [41]. In this contribution, we report on the expression of a designed polycationic, biomineralizing polypeptide, P5S3, by fusing it to ketosteroid isomerase (KSI).
We report the recombinant bacterial expression and purification at high yields of a polycationic oligopeptide, P5S3. The sequence of P5S3 was inspired by a diatom silaffin, a silica precipitating peptide. Like its native model, P5S3 exhibits silica biomineralizing activity, but furthermore has unusual self-assembling properties. P5S3 is efficiently expressed in Escherichia coli as fusion with ketosteroid isomerase (KSI), which causes deposition in inclusion bodies. After breaking the fusion by cyanogen bromide reaction, P5S3 was purified by cation exchange chromatography, taking advantage of the exceptionally high content of basic amino acids. The numerous cationic charges do not prevent, but may even promote counterion-independent self-assembly which in turn leads to silica precipitation. Enzymatic phosphorylation, a common modification in native silica biomineralizing peptides, can be used to modify the precipitation activity.
The secrets of dermcidin action
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In human sweat the most abundant types showing antimicrobial activity are the anionic DCD-1L and DCD-1 (Schittek, 2012; Schittek et al., 2001). These active processed forms kill Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas putida, Salmonella typhimurium) as well as Candida albicans (Cipakova et al., 2006; Lai et al., 2005; Schittek et al., 2001; Steffen et al., 2006; Vuong et al., 2004). Remarkably and untypical for an AMP, the antimicrobial activity of DCD-1(L) is maintained over a broad pH range and at high salt concentrations that resemble the conditions in human sweat (Schittek et al., 2001).
Antimicrobial peptides (AMPs) are important effector molecules of the innate immune defense of diverse species. The majority of known AMPs are cationic therefore facilitating the initial binding of the positively charged peptides to the negatively charged bacterial membrane. Dermcidin (DCD) is constitutively expressed in eccrine sweat glands, secreted into sweat and transported to the epidermal surface where it is proteolytically processed giving rise to several truncated DCD peptides. Its processed forms such as the anionic 48mer DCD-1L and the 47mer DCD-1 possess antimicrobial activity against numerous bacteria including Staphylococcus aureus. Here, the latest knowledge regarding the mode of action of the anionic DCD-1(L) and the functional consequences of their interaction with bacterial membranes is reviewed. There is evidence that the interaction of DCD-1(L) with negatively charged bacterial phospholipids leads to Zn²⁺ dependent formation of oligomeric complexes in the bacterial membrane, which subsequently leads to ion channel formation resulting in membrane depolarization and bacterial cell death.
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Biophysical and structural characterization of G protein-coupled receptors (GPCRs) has been limited due to difficulties in expression, purification, and vitro stability of the full-length receptors. “Divide and conquer” approaches aimed at the NMR characterization of peptides corresponding to specific regions of the receptor have yielded insights into the structure and dynamics of GPCR activation and signaling. Though significant progress has been made in the generation of peptides that are composed of GPCR transmembrane domains, current methods utilize fusion protein strategies that require chemical cleavage and peptide separation via chromatographic means. We have developed an expression and purification system based on fusion to ketosteroid isomerase, thrombin cleavage, and tandem affinity chromatography that enables the solubilization, cleavage, and characterization in a single detergent system relevant for biophysical and structural characterization. We have applied this expression and purification system to the production and characterization of peptides of the adenosine receptor family of GPCRs in Escherichia coli. Herein, we demonstrate using a model peptide that includes extracellular loop 3, transmembrane domain 7, and a portion of the carboxy-terminus of the adenosine A₂a receptor that the peptide is sufficiently pure for biophysical characterization, where it adopts α-helical structure. Furthermore, we demonstrate the utility of this system by optimizing the construct for thrombin processing and apply the system to peptides with more complex structures.
Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat
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Evidence for a clinical relevance of DCD peptides came from our previous studies indicating that patients with atopic dermatitis have a reduced amount of DCD peptides in sweat which contributed to the high susceptibility of these patients to skin infections and to altered bacterial skin colonization (17). The most abundant DCD peptide in sweat is the anionic DCD-1L (48-mer, net charge −2), which is able to kill pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Staphylococcus epidermidis, methicillin-resistant S. aureus, rifampin- and isoniazid-resistant Mycobacterium tuberculosis, Pseudomonas putida, Listeria monocytogenes, Salmonella thyphimurium, and Candida albicans (13, 18–20). Remarkably and untypical for an AMP, the antimicrobial activity of DCD-1L is maintained over a broad pH range and at high salt concentrations that resemble the conditions in human sweat (13).
Dermcidin encodes the anionic amphiphilic peptide DCD-1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD-1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis that is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD-1L forms oligomeric complexes that are stabilized by Zn²⁺. DCD-1L is able to form ion channels in the bacterial membrane, and we propose that Zn²⁺-induced self-assembly of DCD-1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow us for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat.
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Fusion strategies have been successfully employed for the production of a number of cationic peptides using Escherichia coli hosts. Examples include the expression of dermicidin as fusions with ketosteroid isomerase [7], bacteriocin as fusions with GST [8], human β-defensin [9] and adenoregulin [10] as fusions with thioredoxin. Fusion systems are also designed to facilitate purification or to enhance solubility of the target polypeptides [11].
Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni–NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems.

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Protein Expression and Purification

Abstract

Section snippets

Strains and plasmids

Results and discussion

Conclusions

Acknowledgments

References (12)

Detection of dermcidin-derived peptides in sweat by ProteinChip technology

J. Immunol. Methods

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding

Anal. Biochem.

Ultrasensitive assays for endogenous antimicrobial polypeptides

J. Immunol. Methods

High-level expression of soluble human β-defensin-2 in Escherichia coli

Process Biochem.

Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL-37, in Escherichia coli

Protein Express. Purif.

Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide

Biochem. Biophys. Res. Commun.

Cited by (51)

Oral Bifidobacterium longum expressing GLP-2 improves nutrient assimilation and nutritional homeostasis in mice

High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization

The secrets of dermcidin action

An expression and purification system for the biosynthesis of adenosine receptor peptides for biophysical and structural characterization

Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat

Production of recombinant peptides as fusions with SUMO