Abstract:
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 (beta2GPI) 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 microg/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 beta2GPI. 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 beta2GPI 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.
journal_name
BMC Biotechnoljournal_title
BMC biotechnologyauthors
Ioannou Y,Giles I,Lambrianides A,Richardson C,Pearl LH,Latchman DS,Isenberg DA,Rahman Adoi
10.1186/1472-6750-6-8keywords:
subject
Has Abstractpub_date
2006-02-10 00:00:00pages
8issn
1472-6750pii
1472-6750-6-8journal_volume
6pub_type
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