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Title: Encapsulin carrier proteins for enhanced expression of antimicrobial peptides

Abstract

Antimicrobial peptides (AMPs) are regarded as attractive alternatives to conventional antibiotics, but their production in microbes remains challenging due to their inherent bactericidal nature. To address these limitations, we have developed a novel AMP fusion protein system based on an encapsulin nanocompartment protein and have demonstrated its utility in enhancing expression of HBCM2, an AMP with activity against Gram–negative bacteria. Here, HBCM2 was fused to the N–terminus of several Encapsulin monomer (Enc) variants engineered with multiple TEV protease recognition site insertions to facilitate proteolytic release of the fused HBCM2. Fusion of HBCM2 to the Enc variants, but not other common carrier proteins, enabled robust overexpression in Escherichia coli C43(DE3) cells. Interestingly, variants with a TEV site insertion following residue K71 in Enc exhibited the highest overexpression and HBCM2 release efficiencies compared to other variants but were deficient in cage formation. HBCM2 was purified from the highest expressing variant following TEV protease digestion and was found to be highly active in inhibiting E. coli growth (MIC = 5 μg/ml). Our study demonstrates the potential use of the Enc system to enhance expression of AMPs for biomanufacturing and therapeutic applications.

Authors:
 [1];  [1];  [1];  [2]; ORCiD logo [1]
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  2. Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1578250
Report Number(s):
LLNL-JRNL-778977
Journal ID: ISSN 0006-3592; 972185
Grant/Contract Number:  
AC52-07NA27344
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Name: Biotechnology and Bioengineering; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
Biological and medical sciences; antimicrobial peptide; encapsulin; fusion protein; HBCM2; nanocompartment; protein cage; toxic peptide production

Citation Formats

Lee, Tek ‐Hyung, Carpenter, Timothy S., D'haeseleer, Patrik, Savage, David F., and Yung, Mimi C. Encapsulin carrier proteins for enhanced expression of antimicrobial peptides. United States: N. p., 2019. Web. doi:10.1002/bit.27222.
Lee, Tek ‐Hyung, Carpenter, Timothy S., D'haeseleer, Patrik, Savage, David F., & Yung, Mimi C. Encapsulin carrier proteins for enhanced expression of antimicrobial peptides. United States. doi:10.1002/bit.27222.
Lee, Tek ‐Hyung, Carpenter, Timothy S., D'haeseleer, Patrik, Savage, David F., and Yung, Mimi C. Sun . "Encapsulin carrier proteins for enhanced expression of antimicrobial peptides". United States. doi:10.1002/bit.27222.
@article{osti_1578250,
title = {Encapsulin carrier proteins for enhanced expression of antimicrobial peptides},
author = {Lee, Tek ‐Hyung and Carpenter, Timothy S. and D'haeseleer, Patrik and Savage, David F. and Yung, Mimi C.},
abstractNote = {Antimicrobial peptides (AMPs) are regarded as attractive alternatives to conventional antibiotics, but their production in microbes remains challenging due to their inherent bactericidal nature. To address these limitations, we have developed a novel AMP fusion protein system based on an encapsulin nanocompartment protein and have demonstrated its utility in enhancing expression of HBCM2, an AMP with activity against Gram–negative bacteria. Here, HBCM2 was fused to the N–terminus of several Encapsulin monomer (Enc) variants engineered with multiple TEV protease recognition site insertions to facilitate proteolytic release of the fused HBCM2. Fusion of HBCM2 to the Enc variants, but not other common carrier proteins, enabled robust overexpression in Escherichia coli C43(DE3) cells. Interestingly, variants with a TEV site insertion following residue K71 in Enc exhibited the highest overexpression and HBCM2 release efficiencies compared to other variants but were deficient in cage formation. HBCM2 was purified from the highest expressing variant following TEV protease digestion and was found to be highly active in inhibiting E. coli growth (MIC = 5 μg/ml). Our study demonstrates the potential use of the Enc system to enhance expression of AMPs for biomanufacturing and therapeutic applications.},
doi = {10.1002/bit.27222},
journal = {Biotechnology and Bioengineering},
number = ,
volume = ,
place = {United States},
year = {2019},
month = {11}
}

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