Soluble expression and purification of human β-defensin DEFB136 in Escherichia coli and identification of its bioactivity

https://doi.org/10.1016/j.pep.2021.105968Get rights and content

Highlights

  • The human beta-defensin DEFB136 peptide was successfully purified from E. coli using pTWIN 1 expression system.

  • The recombinant DEFB136 peptide exhibited a broad spectrum of antimicrobial activity but low cytotoxicity to human RBC.

  • The novel DEFB136 peptide possessed excellent activities manifested in strong LPS-binding capacity.

Abstract

Human β-defensins are an important family of innate host defense peptides with pleiotropic activities. Human β-defensin 36 (DEFB136) is a novel member of the β-defensin family which have not been characterized so far. In the present research, the DEFB136 peptide was expressed successfully and purified using the IMPACT-TWIN 1 expression system. The purified DEFB136 peptide was identified by MALDI-TOF mass spectrometry and circular dichroism spectroscopy. While the recombinant DEFB136 peptide exhibited a broad spectrum of antimicrobial activity against E. coli, Staphylococcus aureus and Candida albicans strains, but had low cytotoxicity to human erythrocytes. In addition, the result of the octet assay showed that the DEFB136 had a high lipopolysaccharide (LPS)-binding affinity, suggesting the DEFB136 may be involved in immunoregulation through its LPS neutralization. These results may help lay the groundwork to understand better the complex interaction between innate host defense and the diversity of the defensin family.

Introduction

The human β-defensin family, as an important part of host innate immunity, is receiving significant attention thanks to its antimicrobial activity and immunoregulation function. Human β-defensins have a structural core of three anti-parallel β-sheets secured by disulfide linkages. These disulfide bridges allow the primary structure of a peptide to be short and diverse [1]. Since the first β-defensin hBD-1 was isolated from the plasma of patients with kidney disease, 39 human β-defensin genes have been identified [2,3]. Human β-defensins are produced primarily in the respiratory, gastrointestinal, as well as genitourinary tracts [4]. Surprisingly, most β-defensin transcripts are preferentially expressed in the male reproductive tract, particularly in epididymis and testis [3,5]. Existence of such a large array of defensins in various segments of the male reproductive tract implies that these defensins may play a dual role in both fertility and host defense [6,7]. To date, however, only the first four human β-defensins have been characterized in some detail.

The cloning and expression of different human β-defensin family proteins may provide new insights into the relationship between the structure, function and specificity of antimicrobial peptides. We have expressed and characterized a variety of active human β-defensins located in the epididymis [[8], [9], [10], [11]], which included a novel β-defensin DEFB136. In addition, the DEFB136 was also found on mucosal surfaces lining the respirator and gastrointestinal tracts, as well as on the skin. However, RT-PCR revealed that the DEFB136 transcripts are preferentially expressed in the male reproductive tract. Defb136 gene (GenBank: DQ012026.1) was located on chromosome 8p23.1 [3]. Copy number variation (CNV) studies showed that the human β-defensin cluster at the human region 8p23.1 has potential clinical relevance for innate immunity, inflammation, and cancer [12,13]. However, few pieces of information were known about DEFB136 so far. It is necessary to produce enough protein by recombinant expression to obtain more structural and functional information of defb136.

The full-length DEFB136 is composed of 78 amino acids, and the mature peptide contains 57 amino acids. In this study, the recombinant DEFB136 peptide was expressed using the pTWIN1 expression system in E. coli BL21 (DE3) cells and was well characterized. To investigate the general biological properties of the DEFB136 peptides, their antimicrobial and LPS-binding activities were also determined. Our results may contribute to understanding the complex interaction between DEFB136 peptide and other human defensins in the male reproductive tract.

Section snippets

Strains and plasmids

E. coli DH5α (maintained in our laboratory) was used as host strain for cloning and plasmid amplification. E. coli BL21 (DE3) (Novagen, USA) was used as host strain for expression. The vector pTWIN1 (New England Biolabs, Inc., Beijing, China) was used to construct expression vector. E. coli K12D31, Staphylococcus aureus CMCC 26003 and Candida albicans SC 5314 (maintained in our laboratory) were used for antimicrobial assays.

Construction of the expression vector

The defb136 cDNA sequence was subcloned by PCR from the pGEM-DEFB136

Construction of pTWIN1-DEFB136 expression vector

DEFB136 sequences consist of a characteristic signal sequence and a conserved defensin motif. In this work, the DEFB136 mature peptide as a novel β-defensin was prepared to investigate its structural and functional properties. The structure of the expression plasmid pTWIN1-DEFB136 is shown in Fig. 1. The nucleotide sequence encoding DEFB136 was inserted into the C-terminus of Ssp DnaB intein of plasmid pTWIN 1 (Fig. 1b). The amino acid sequence of the DEFB136 mature peptide shown in Fig. 1a was

Conclusion

In this study, the soluble DEFB136 peptide was expressed in E. coli using the pTWIN 1 expression system, which is the first report of the production and purification of bioactive DEFB136. The DEFB136 peptide, as a novel member of the human β-defensins family, maintained a conserved β-sheet domain of the β-defensin family. In addition, the recombinant DEFB136 peptide also exhibited a broad spectrum of antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans

CRediT authorship contribution statement

Haiyan Liu: Methodology, Investigation, Formal analysis, Writing–original draft, Funding acquisition. Hua Diao: Investigation, Writing–review & editing, Data curation, Supervision. Jing Hou: Writing–review & editing, Funding acquisition, Project administration. Heguo Yu: Investigation, Data curation. Huiping Wen: Software.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by Key Science and Technology Innovation Team Funding Project of Lishui city, China (2018CXTD02), Key Research and Development Program of Lishui city, China (2021ZDYF07).

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    These authors contributed equally to this work.

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