The Small Metal-Binding Protein SmbP Simplifies the Recombinant Expression and Purification of the Antimicrobial Peptide LL-37.

David A Perez-Perez,Nestor G Casillas-Vega,Teresa De J Villanueva-Ramirez,Patricia Gonzalez-Barranco,Xristo Zarate,Adriana E Hernandez-Pedraza

doi:10.3390/antibiotics10101271

David A Perez-Perez, Nestor G Casillas-Vega + Show 4 more

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https://doi.org/10.3390/antibiotics10101271

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Abstract

(1) Background: The cathelicidin peptide LL-37 is a prominent molecule with many biological activities, including antimicrobial. Due to its importance, here, we describe the production of LL-37 tagged with SmbP, a relatively new carrier protein that improves the production of recombinant proteins and peptides in Escherichia coli. We present an alternative method for the rapid expression, purification, and antimicrobial evaluation of LL-37, that involves only one purification step. (2) Methods: A DNA construct of SmbP_LL-37 was transformed into E. coli BL21(DE3); after overnight expression, the protein was purified directly from the cell lysate using immobilized metal-affinity chromatography. SmbP_LL-37 was treated with Enterokinase to obtain the free LL-37 peptide. The antimicrobial activity of both SmbP_LL-37 and free LL-37 was determined using the colony forming unit assay method. (3) Results: SmbP_LL-37 was observed in the soluble fraction of the cell lysate; after purification with IMAC, protein gel electrophoresis, and analysis by ImageJ, it showed 90% purity. A total of 3.6 mg of SmbP_LL-37 was produced from one liter of cell culture. SmbP_LL-37 and free LL-37 both showed inhibition activity against Staphylococcus aureus and Escherichia coli. (4) Conclusions: The SmbP fusion protein is a valuable tool for producing biologically-active LL-37 peptide. The production method described here should be of interest for the expression and purification of additional cationic peptides, since it cuts the purification time considerably prior to determination of antimicrobial activity.

Highlights

The small metal-binding protein SmbP isolated from the periplasm of the bacterium
Via its metal-binding capacity, recombinant proteins can be purified through immobilized metal-affinity chromatography (IMAC), obtaining high purities in a single step [2,3]
We have previously reported the production of biologically active proteins, such as the human growth hormone, and the antimicrobial peptides (AMPs) Bin1b and VpDef tagged with SmbP, with satisfactory yields and purities [4,5,6]

Summary

Introduction

The small metal-binding protein SmbP isolated from the periplasm of the bacterium. Nitrosomonas europaea is an attractive fusion protein for production of recombinant proteins and peptides in Escherichia coli. It is a 9.9-kDa protein whose biological function is the expulsion of toxic metal ions from the cell [1]. SmbP improves the expression of recombinant proteins and peptides by increasing their solubility and through avoiding the formation of inclusion bodies. Via its metal-binding capacity, recombinant proteins can be purified through immobilized metal-affinity chromatography (IMAC), obtaining high purities in a single step [2,3]. Proteins are typically digested with the protease Enterokinase to remove SmbP, obtaining the pure target protein or peptide after a

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