Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag

The increasing demand for antigenic peptides in the development of novel serologic diagnostics and epitope-based vaccines requires rapid and reliable peptide synthesis techniques. Here we investigated a method for efficient recombinant expression and purification of medium- to large-sized antigenic peptides in E. coli. Previously we devised a streamlined protein expression and purification scheme based on a cleavable self-aggregating tag (cSAT), which comprised an intein molecule and a self-aggregating peptide ELK16. In this scheme, the target proteins were fused in the C-termini with cSAT and expressed as insoluble aggregates. After intein self-cleavage, target proteins were released into the soluble fraction with high yield and reasonable purity. We demonstrated the applicability of this scheme by preparing seven model viral peptides, with lengths ranging from 32 aa to 72 aa. By adding an N-terminal thioredoxin tag, we enhanced the yield of target peptides released from the aggregates. The purified viral peptides demonstrated high antigenic activities in ELISA and were successfully applied to dissecting the antigenic regions of influenza hemagglutinin. The cSAT scheme described here allows for the rapid and low-cost preparation of multiple antigenic peptides for immunological screening of a broad range of viral antigens.

[1]  Zirong Wu,et al.  Expression and purification of Canis interferon α in Escherichia coli using different tags. , 2015, Protein expression and purification.

[2]  Gerald Striedner,et al.  Npro fusion technology to produce proteins with authentic N termini in E. coli , 2007, Nature Methods.

[3]  L. Domingues,et al.  Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system , 2014, Front. Microbiol..

[4]  Pier Luigi Lopalco,et al.  Are the Two Human Papillomavirus Vaccines Really Similar? A Systematic Review of Available Evidence: Efficacy of the Two Vaccines against HPV , 2015, Journal of immunology research.

[5]  R. Valenta,et al.  Vaccine development for allergen-specific immunotherapy based on recombinant allergens and synthetic allergen peptides: Lessons from the past and novel mechanisms of action for the future , 2016, The Journal of allergy and clinical immunology.

[6]  B. Bray.  Large-scale manufacture of peptide therapeutics by chemical synthesis , 2003, Nature Reviews Drug Discovery.

[7]  J. R. Christensen,et al.  Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation. , 2015, Journal of controlled release : official journal of the Controlled Release Society.

[8]  Zhanglin Lin,et al.  Screening of Random Peptide Library of Hemagglutinin from Pandemic 2009 A(H1N1) Influenza Virus Reveals Unexpected Antigenically Important Regions , 2011, PloS one.

[9]  Alan S. Perelson,et al.  High Multiplicity Infection by HIV-1 in Men Who Have Sex with Men , 2010, PLoS pathogens.

[10]  S. Rüdiger,et al.  Studying protein-protein interactions using peptide arrays. , 2011, Chemical Society reviews.

[11]  Roland Benz,et al.  Clostridium perfringens Delta Toxin Is Sequence Related to Beta Toxin, NetB, and Staphylococcus Pore-Forming Toxins, but Shows Functional Differences , 2008, PloS one.

[12]  Sjoerd H van der Burg,et al.  Design and development of synthetic peptide vaccines: past, present and future , 2007, Expert review of vaccines.

[13]  Identification of Viral Peptide Fragments for Vaccine Development , 2009, Methods in molecular biology.

[14]  M. Bottomley,et al.  Structural and Computational Biology in the Design of Immunogenic Vaccine Antigens , 2015, Journal of immunology research.

[15]  E. P. Hudson,et al.  Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays* , 2014, Molecular & Cellular Proteomics.

[16]  Stefan Rose-John,et al.  Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. , 2010, Trends in biotechnology.

[17]  Liang Feng,et al.  Simple bioseparations using self-cleaving elastin-like polypeptide tags , 2005, Nature Methods.

[18]  Zhanglin Lin,et al.  Streamlined protein expression and purification using cleavable self-aggregating tags , 2011, Microbial cell factories.

[19]  Karl A. Hassan,et al.  Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module , 2011, PloS one.

[20]  G. Evan,et al.  Synthetic peptides in biochemical research , 1995, Molecular biotechnology.

[21]  Zhanglin Lin,et al.  Aggregating tags for column‐free protein purification , 2015, Biotechnology journal.

[22]  Jinpeng Yu,et al.  Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli , 2016, Marine drugs.

[23]  Jie Feng,et al.  Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli. , 2014, Canadian journal of microbiology.

[24]  D. Holdstock.  Past, present--and future? , 2005, Medicine, conflict, and survival.

[25]  M. Humbert,et al.  Inducing Cross-Clade Neutralizing Antibodies against HIV-1 by Immunofocusing , 2008, PloS one.

[26]  S. Ishii,et al.  Increase of Solubility of Foreign Proteins in Escherichia coli by Coproduction of the Bacterial Thioredoxin (*) , 1995, The Journal of Biological Chemistry.

[27]  A. Székács,et al.  Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners. , 2015, Journal of microbiological methods.

[28]  J. Mccoy,et al.  A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm , 1993, Bio/Technology.

[29]  Zhanglin Lin,et al.  Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli , 2011, Microbial cell factories.

[30]  R. Hai,et al.  Broadly Protective Monoclonal Antibodies against H3 Influenza Viruses following Sequential Immunization with Different Hemagglutinins , 2010, PLoS pathogens.

[31]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[32]  Erika Salvi,et al.  Adducin- and Ouabain-Related Gene Variants Predict the Antihypertensive Activity of Rostafuroxin, Part 2: Clinical Studies , 2010, Science Translational Medicine.

[33]  Yibing Huang,et al.  Prokaryotic expression and mechanism of action of α-helical antimicrobial peptide A20L using fusion tags , 2015, BMC Biotechnology.

[34]  Xuegang Luo,et al.  High-Level Expression of the Antimicrobial Peptide Plectasin in Escherichia coli , 2010, Current Microbiology.

[35]  S. Stephenson,et al.  Evaluation of Physarum polycephalum plasmodial growth and lipid production using rice bran as a carbon source , 2015, BMC Biotechnology.

[36]  L. Mangamoori,et al.  Recombinant approach for the production of HIV fusion inhibitor Enfuvirtide using Escherichia coli. , 2014, Protein expression and purification.

[37]  A. Kajava,et al.  Long Synthetic Peptides for the Production of Vaccines and Drugs: A Technological Platform Coming of Age , 2010, Science Translational Medicine.

[38]  B. Sykes,et al.  Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli , 2014, FEBS letters.

[39]  D. Ekiert,et al.  Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes , 2010, Proceedings of the National Academy of Sciences.

[40]  R. Robinson,et al.  Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein , 2014, Molecular Biology Reports.