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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Mycotoxin Prevention and Applied Microbiology Research » Research » Publications at this Location » Publication #315861
Title: A combined strategy improving the expression and solubility of deoxynivalenol single-chain variable fragment antibodies

Author
item SANDERS, MELANIE - Orise Fellow
item MURPHY, C - Dublin City University
item O'KENNEDY, R - Dublin City University
item DE SAEGER, S - Ghent University
item Maragos, Chris

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/8/2015
Publication Date: 5/8/2015
Citation: Sanders, M., Murphy, C.S., O'Kennedy, R., De Saeger, S., Maragos, C.M. 2015. A combined strategy improving the expression and solubility of deoxynivalenol single-chain variable fragment antibodies [abstract].

Interpretive Summary:

Technical Abstract: Deoxynivalenol (DON) is a secondary metabolite produced by certain species of fungi, in particular Fusarium graminearum and F. culmorum. These fungi can infest small grains causing significant economic damage to cereal crops worldwide. Animal exposure to DON can lead to reduced food or feed consumption, abdominal distress, malaise, diarrhoea, shock, and death in extremely high DON doses. To protect human and animal health, many countries have established regulatory levels for DON in grains. In the United States the advisory level is 1 mg DON/kg (1 ppm) and in Europe maximum levels between 200 and 1750 ug DON/kg are advised by the European Commission. To assure high quality grain is used in food and feedstuffs, many analytical techniques have been applied for the detection and quantitation of DON. The development of antibody-based technologies is advancing rapidly and has resulted in many novel applications, such as surface-plasmon based immunoassays, flow cytometry, and biolayer interferometry. Due to the higher affinity and solubility of intact antibodies and the existence of problems expressing fragments in Escherichia coli (E. coli), intact antibodies are more commonly used. As recombinant antibodies such as single-chain variable fragments (scFv) are easily manipulated and genetically modified, a solution is desired for the aforementioned problems. First, we compared two different sizes of the glycine serine linker between the variable heavy (VH) and variable light (VL) chain, because this determines the formation of antibody monomers, dimmers, and trimers and therefore influences target affinity. Also, the use of different phagemid expression vectors was evaluated. The classic pComb3XSS was compared to a pET expression system, characterized by its high-level transcription and translation. The phagemids were transferred into XL-1 Blue E. coli cell strains, One Shot® TOP 10 chemically competent cells or OrigamiTM 2 competent cells to evaluate them for oxidizing versus reducing conditions and periplasmic versus cytoplasmic expression of the antibody. Finally, the transferred E. coli cell lines were co-cultured with plasmids expressing the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone teams in order to facilitate assessment of the effects of these chaperone teams on folding or assembly of the recombinant proteins. For the evaluation, three different DON scFv antibodies were used. The results will be presented during the poster presentation.