skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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 Volume: 117; Journal Issue: 3; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL 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:https://doi.org/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:https://doi.org/10.1002/bit.27222. https://www.osti.gov/servlets/purl/1578250.
@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 = 3,
volume = 117,
place = {United States},
year = {2019},
month = {11}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3)
journal, February 2009

  • Tian, Zi-gang; Dong, Tian-tang; Yang, Ya-lin
  • Applied Microbiology and Biotechnology, Vol. 83, Issue 1
  • DOI: 10.1007/s00253-009-1893-z

Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin
journal, June 2016


Tuning Escherichia coli for membrane protein overexpression
journal, September 2008

  • Wagner, S.; Klepsch, M. M.; Schlegel, S.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 38
  • DOI: 10.1073/pnas.0804090105

High-Level Expression of Antimicrobial Peptide Mediated by a Fusion Partner Reinforcing Formation of Inclusion Bodies
journal, November 2000

  • Lee, J. H.; Kim, J. H.; Hwang, S. W.
  • Biochemical and Biophysical Research Communications, Vol. 277, Issue 3
  • DOI: 10.1006/bbrc.2000.3712

Structural basis of enzyme encapsulation into a bacterial nanocompartment
journal, August 2008

  • Sutter, Markus; Boehringer, Daniel; Gutmann, Sascha
  • Nature Structural & Molecular Biology, Vol. 15, Issue 9
  • DOI: 10.1038/nsmb.1473

The co-evolution of host cationic antimicrobial peptides and microbial resistance
journal, June 2006

  • Peschel, Andreas; Sahl, Hans-Georg
  • Nature Reviews Microbiology, Vol. 4, Issue 7
  • DOI: 10.1038/nrmicro1441

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates
journal, January 2012

  • Hong, Paula; Koza, Stephan; Bouvier, Edouard S. P.
  • Journal of Liquid Chromatography & Related Technologies, Vol. 35, Issue 20
  • DOI: 10.1080/10826076.2012.743724

Clustal W and Clustal X version 2.0
journal, September 2007


Using the Basic Local Alignment Search Tool (BLAST)
journal, July 2007

  • Mount, David W.
  • Cold Spring Harbor Protocols, Vol. 2007, Issue 7
  • DOI: 10.1101/pdb.top17

Recombinant production of antimicrobial peptides in Escherichia coli: A review
journal, December 2011


Host defense antimicrobial peptides as antibiotics: design and application strategies
journal, June 2017


Encapsulins: molecular biology of the shell
journal, June 2017

  • Nichols, Robert J.; Cassidy-Amstutz, Caleb; Chaijarasphong, Thawatchai
  • Critical Reviews in Biochemistry and Molecular Biology, Vol. 52, Issue 5
  • DOI: 10.1080/10409238.2017.1337709

Large protein organelles form a new iron sequestration system with high storage capacity
journal, July 2019

  • Giessen, Tobias W.; Orlando, Benjamin J.; Verdegaal, Andrew A.
  • eLife, Vol. 8
  • DOI: 10.7554/eLife.46070

Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform
journal, September 2014

  • Moon, Hyojin; Lee, Jisu; Min, Junseon
  • Biomacromolecules, Vol. 15, Issue 10
  • DOI: 10.1021/bm501066m

Expression systems for heterologous production of antimicrobial peptides
journal, December 2012


An overview of enzymatic reagents for the removal of affinity tags
journal, December 2011


Yeast-Based Synthetic Biology Platform for Antimicrobial Peptide Production
journal, January 2018


Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models
journal, April 2017

  • Hwang, In Young; Koh, Elvin; Wong, Adison
  • Nature Communications, Vol. 8, Issue 1
  • DOI: 10.1038/ncomms15028

Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System
journal, August 2016

  • Ishida, Hiroaki; Nguyen, Leonard T.; Gopal, Ramamourthy
  • Journal of the American Chemical Society, Vol. 138, Issue 35
  • DOI: 10.1021/jacs.6b06781

Recombinant proteins fused to thermostable partners can be purified by heat incubation
journal, January 2004


Packaging guest proteins into the encapsulin nanocompartment from Rhodococcus erythropolis N771 : Packaging Guest Proteins into the Encapsulin Nanocompartment
journal, September 2014

  • Tamura, Akio; Fukutani, Yosuke; Takami, Taku
  • Biotechnology and Bioengineering, Vol. 112, Issue 1
  • DOI: 10.1002/bit.25322

Antimicrobial Peptide Therapeutics for Cystic Fibrosis
journal, June 2005


Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700
journal, January 2017

  • Ji, Shengyue; Li, Weili; Baloch, Abdul Rasheed
  • Scientific Reports, Vol. 7, Issue 1
  • DOI: 10.1038/srep40587

The expanding scope of antimicrobial peptide structures and their modes of action
journal, September 2011


Protein aggregation in bacteria: the thin boundary between functionality and toxicity
journal, September 2013

  • Bednarska, Natalia G.; Schymkowitz, Joost; Rousseau, Frederic
  • Microbiology, Vol. 159, Issue Pt_9
  • DOI: 10.1099/mic.0.069575-0

Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria
journal, July 2013

  • Volzing, Katherine; Borrero, Juan; Sadowsky, Michael J.
  • ACS Synthetic Biology, Vol. 2, Issue 11
  • DOI: 10.1021/sb4000367

Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science
journal, October 2016


Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides
journal, November 2018


Antibacterial peptides for therapeutic use: obstacles and realistic outlook
journal, October 2006


Tailored delivery of analgesic ziconotide across a blood brain barrier model using viral nanocontainers
journal, August 2015

  • Anand, Prachi; O’Neil, Alison; Lin, Emily
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep12497

A designed bacterial microcompartment shell with tunable composition and precision cargo loading
journal, July 2019


APD3: the antimicrobial peptide database as a tool for research and education
journal, November 2015

  • Wang, Guangshun; Li, Xia; Wang, Zhe
  • Nucleic Acids Research, Vol. 44, Issue D1
  • DOI: 10.1093/nar/gkv1278

ConSeq: the identification of functionally and structurally important residues in protein sequences
journal, February 2004


Prokaryotic nanocompartments form synthetic organelles in a eukaryote
journal, April 2018


Short Proline-Rich Antimicrobial Peptides Inhibit Either the Bacterial 70S Ribosome or the Assembly of its Large 50S Subunit
journal, October 2015


Widespread distribution of encapsulin nanocompartments reveals functional diversity
journal, March 2017