Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep High level expression and immunochemical characterization of botulinum neurotoxin type F light chain Ritika Chauhan a , Vinita Chauhan a , Mula Kameshwar Rao b , Dilip Chaudhary c , Sameer Bhagyawant d , Ram Kumar Dhaked a,* a Biotechnology Division, India b Division of Pharmacology and Toxicology, Defence Research & Development Establishment, Jhansi Road, Gwalior 474002, India c Defence Laboratory, Ratanada, Jodhpur 342011, India d School of Studies in Biotechnology, Jiwaji University, Gwalior 474001, India ARTICLE INFO Keywords: Clostridium botulinum Botulinum neurotoxins type F (BoNT/F) Surface plasmon resonance (SPR) Vesicle associated membrane protein (VAMP-2) Small molecule inhibitors (SMIs) ABSTRACT Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F- LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based de- tection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent de- tection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis. 1. Introduction The botulinum neurotoxins (BoNTs) are the endopeptidases pro- duced by Gram positive, rod shaped, motile, non-encapsulated, spore forming anaerobic bacteria Clostridium botulinum, C. butyricum and C. baratii as ∼150 kDa single polypeptide chain [1,2]. These neurotoxins are the most toxic substances known to humankind and have been evidently related to neuroparalytic illness ‘botulism’, which could prove fatal in course of time owing to respiratory failure [3]. Instead of rare natural occurrence of botulism, the botulinum neurotoxins have been classified as Category A biological threat agents by Centers for Disease Control and Prevention (CDC), USA [4], considering the ease in pro- duction and dissemination, unavailability of reliable treatment and limited supportive care facility [5]. There are seven antigenically dis- tinct classes denoted BoNT/A-G [2] and very recently discovered yet debatable eighth serotype BoNT/H [6]. One of the discoveries re- cognized it as a novel serotype while further study proved it as subtype of pre-existing serotypes [7]. Even though the reported human cases are of serotypes A, B, E, and F; interestingly, all the serotypes may poten- tially infect human. All toxinotypes inhibit acetylcholine release from nerve terminals but vary significantly in their binding receptors and intracellular target proteins [8,9]. These toxins are proteolytic cleaved into dichain consisting of a ∼100 kDa heavy chain (HC) and a ∼50 kDa light chain (LC) that re- main associated in native state by disulfide linkage. The ∼100 kDa HC is composed of the cell receptor binding carboxy terminal (H C ) and translocation amino terminal (H N ) domains and the ∼50 kDa LC act as catalytic domain. The H C binds to the receptor present on the un- myelinated presynaptic membrane of the cholinergic cells leading to the internalization of the neurotoxin by endocytosis. The translocation of the LC takes place through a channel arbitrated by the H N domain from the endosome to the cell cytosol [10]. The Zn endopeptidase ac- tivity of the LC leads to the proteolytic cleavage of either of the Soluble N-ethylmaleimide sensitive factor Attachment Receptor (SNARE) https://doi.org/10.1016/j.pep.2018.01.014 Received 1 December 2017; Received in revised form 16 January 2018; Accepted 27 January 2018 * Corresponding author. E-mail address: rkdhaked@drde.drdo.in (R.K. Dhaked). Protein Expression and Purification 146 (2018) 51–60 Available online 31 January 2018 1046-5928/ © 2018 Elsevier Inc. All rights reserved. T