Abstract
Recombinant antibodies are used with great success in many different diagnostic and therapeutic applications. A variety of protein expression systems are available, but nowadays almost all therapeutic antibodies are produced in mammalian cell lines due to their complex structure and glycosylation requirements. However, production of clinical-grade antibodies in mammalian cells is very expensive and time-consuming. On the other hand, Escherichia coli (E. coli) is known to be the simplest, fastest and most cost-effective recombinant expression system, which usually achieves higher protein yields than mammalian cells. Indeed, it is one of the most popular host in the industry for the expression of recombinant proteins. In this work, a trivalent single-chain fragment variable (scFv)-based N-terminal trimerbody, specific for native laminin-111, was expressed in human embryonic kidney 293 cells and in E. coli. Mammalian and bacterially produced anti-laminin trimerbody molecules display comparable functional and structural properties, although importantly the yield of trimerbody expressed in E. coli was considerably higher than in human cells. These results demonstrated that E. coli is a versatile and efficient expression system for multivalent trimerbody-based molecules that is suitable for their industrial production.
Highlights
Recombinant antibodies represent one of the fastest growing class of biopharmaceutical products and are established as important tools for research, diagnosis and therapy (Leavy 2010)
Our results demonstrate that E. coli is a viable alternative expression system for single-chain fragment variable (scFv)-based N-terminal trimerbody molecules
The induction of antibody expression was achieved with a combination of low temperature, reduced IPTG concentration (0.1 mM) and long induction times, and the presence and functionality of L36 scFv and L36 scFv-based N-terminal trimerbody (L36N) in the periplasmic fraction was studied by western blot and ELISA
Summary
Recombinant antibodies represent one of the fastest growing class of biopharmaceutical products and are established as important tools for research, diagnosis and therapy (Leavy 2010). We have recently reported the in vitro and in vivo properties of multivalent antibodies generated by fusing a trimerization (TIE) domain to the N- or C-terminus of a single-chain variable fragment (scFv) (Sánchez-Arevalo Lobo et al 2006; Cuesta et al 2009, 2012; Blanco-Toribio et al 2013). TIE domains are composed of the N-terminal trimerization region of collagen XVIII NC1 (TIEXVIII) or collagen XV NC1 (TIEXV) flanked by flexible linkers. This new antibody format, termed trimerbody, is trimeric in solution and exhibited excellent antigen binding capacity and multivalency (Sánchez-Arevalo Lobo et al. Blanco‐Toribio et al AMB Expr (2015) 5:45
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