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A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli
BACKGROUND: The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β(2)GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (a...
Autores principales: | , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2006
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1402286/ https://www.ncbi.nlm.nih.gov/pubmed/16472380 http://dx.doi.org/10.1186/1472-6750-6-8 |
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author | Ioannou, Yiannis Giles, Ian Lambrianides, Anastasia Richardson, Chris Pearl, Laurence H Latchman, David S Isenberg, David A Rahman, Anisur |
author_facet | Ioannou, Yiannis Giles, Ian Lambrianides, Anastasia Richardson, Chris Pearl, Laurence H Latchman, David S Isenberg, David A Rahman, Anisur |
author_sort | Ioannou, Yiannis |
collection | PubMed |
description | BACKGROUND: The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β(2)GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. METHODS: Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. RESULTS: Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β(2)GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. CONCLUSION: By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of β(2)GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production. |
format | Text |
id | pubmed-1402286 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-14022862006-03-16 A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli Ioannou, Yiannis Giles, Ian Lambrianides, Anastasia Richardson, Chris Pearl, Laurence H Latchman, David S Isenberg, David A Rahman, Anisur BMC Biotechnol Methodology Article BACKGROUND: The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β(2)GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. METHODS: Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. RESULTS: Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β(2)GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. CONCLUSION: By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of β(2)GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production. BioMed Central 2006-02-10 /pmc/articles/PMC1402286/ /pubmed/16472380 http://dx.doi.org/10.1186/1472-6750-6-8 Text en Copyright © 2006 Ioannou et al; licensee BioMed Central Ltd. |
spellingShingle | Methodology Article Ioannou, Yiannis Giles, Ian Lambrianides, Anastasia Richardson, Chris Pearl, Laurence H Latchman, David S Isenberg, David A Rahman, Anisur A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli |
title | A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli |
title_full | A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli |
title_fullStr | A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli |
title_full_unstemmed | A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli |
title_short | A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli |
title_sort | novel expression system of domain i of human beta2 glycoprotein i in escherichia coli |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1402286/ https://www.ncbi.nlm.nih.gov/pubmed/16472380 http://dx.doi.org/10.1186/1472-6750-6-8 |
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