Protein Expression and PuriWcation xxx (2006) xxx–xxx www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.08.009 ARTICLE IN PRESS Please cite this article as: Xuejun Hu et al., Optimisation of production of a domoic acid-binding scFv antibody fragment in Escheri- chia coli using molecular chaperones and functional . . . , Protein Expression and PuriWcation (2006), doi:10.1016/j.pep.2006.08.009 Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support Xuejun Hu a , Liam O’Hara b , Simon White a,c , Edmond Magner a,c , Marian Kane b , J. Gerard Wall a,c,¤ a Department of Chemical and Environmental Sciences, University of Limerick, Plassey Technology Park, Limerick, Ireland b National Diagnostic Centre, National University of Ireland, Galway, Ireland c Materials and Surface Science Institute, University of Limerick, Plassey Technology Park, Limerick, Ireland Received 8 August 2006, and in revised form 14 August 2006 Abstract Domoic acid is a potent neurotoxin that can lead to amnesic shellWsh poisoning in humans through ingestion of contaminated shell- Wsh. We have produced and puriWed an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaper- ones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulWde bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this eVect was not synergistic with molecular chaperone co-production. Mean- while, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was pre- dominantly in insoluble form and could not be rescued by chaperones. PuriWcation of the scFv was achieved using an optimised metal aYnity chromatography procedure and the puriWed protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential beneWt of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development.  2006 Elsevier Inc. All rights reserved. Keywords: Escherichia coli; Recombinant antibody; scFv; Chaperone; Domoic acid; Mesoporous silicate; Immunosensor Domoic acid is a naturally occurring neurotoxin pro- duced by species of the diatom genus Pseudo-nitzschia and concentrated in shellWsh that ingest the algal species. It leads to amnesic shellWsh poisoning (ASP) 1 in humans, which is characterised by vomiting and diarrhoea in mild cases but can lead to neurological symptoms and possibly death in more severe attacks. As there is no antidote for ASP, on-site testing of shellWsh samples is extremely impor- tant in limiting human exposure. Current tests for domoic acid are based on analytical methods such as HPLC [1] and liquid chromatography with tandem mass spectrometry [2] or mouse toxicity assays [3], none of which is suitable for rapid, on-site testing or high throughput screening of samples. Antibody molecules can be produced in Escherichia coli in the form of small, recombinant fragments that retain the binding properties of their parent monoclonal antibodies [4]. This is a more economic and faster process than mono- clonal antibody production in mammalian expression sys- tems. In addition, the smaller size of the fragments may also * Corresponding author. Fax: +353 61 202568. E-mail address: gerard.wall@ul.ie (J. Gerard Wall). 1 Abbreviations used: ASP, amnesic shellWsh poisoning; TEE, transla- tion enhancer element; TF, trigger factor; scFv, single-chain Fragment variable.