Abstract
BackgroundIn this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (β2GPI). β2GPI is intrinsic to the pathological progression of the Antiphospholipid Syndrome (APS). Patients develop autoantibodies targeting an epitope located on the N-terminal Domain I of β2GPI rendering this domain of interest as a possible therapeutic.ResultsThis new method of production of Domain I of β2GPI has increased the production yield by ~20 fold compared to previous methods in E.coli. This largely scalable, partially automated method produces 50–75 mg of pure, folded, active Domain I of β2GPI per litre of expression media.ConclusionThe application of this method may enable production of Domain I on sufficient scale to allow its use as a therapeutic.
Highlights
In this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (β2GPI). β2GPI is intrinsic to the pathological progression of the Antiphospholipid Syndrome (APS)
In this paper we describe the development of a novel method for medium scale bacterial expression of a small protein domain with considerable therapeutic potential, the N-terminal domain of beta-2-glycoprotein I (β2GPI), commonly designated domain I (DI)
The gel was run, washed briefly in ddH2O and stained for 1 h with InstantBlueTM stain (Expedeon, UK). b & c Densitometric analysis was carried out on Luria Broth (LB) and Terrific Broth (TB) samples on a small scale loaded in an identical way, gels were scanned, images converted to TIFF files and analysed
Summary
In this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (β2GPI). β2GPI is intrinsic to the pathological progression of the Antiphospholipid Syndrome (APS). In this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (β2GPI). Protein-based biologic agents are increasingly used in the treatment of a range of autoimmune diseases They may exert their effects by blocking receptor-ligand or antibody-antigen interactions or by delivering an exogenous enzyme such as uricase [1,2,3,4,5]. Refolding aggregated protein is a complex process resulting in low yields [15, 16] Another limitation of expression in the bacterial cytoplasm is the impossibility of obtaining proteins with post-translational
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