Expression of antibody fragments by periplasmic secretion in Escherichia coli.
نویسندگان
چکیده
Antibody-based drugs are increasingly used in the clinic, and their importance is set to escalate in the coming years as more drugs in this class progress through clinical trials. Although many such drugs utilize whole antibodies, others exploit fragments, e.g., fragment antigen binding (Fab') or single-chain fragment variable (scFv), which retain the antigen-binding specificity without the fragment crystallizable (Fc) element necessary to mediate effector functions. Antibody fragments can be advantageous for many therapeutic uses, owing to the fact that valency and half-life can be tailored through protein engineering approaches to suit the desired mechanism of action (1). Furthermore, antibody fragments are more suited to expression in microbial systems, providing benefits in terms of increased scale and ease of manufacture (2). Escherichia coli is currently the host of choice for producing antibody fragments. For disulfide-bonded proteins (Fab' and scFv), good expression levels have been achieved via soluble production, secreting the Fab' chains into the oxidizing environment of the bacterial periplasm where assembly and disulfide bond formation can occur. Periplasmic secretion is achieved by genetically fusing the signal sequence from an E. coli protein onto the N-terminus of the antibody V-region sequence. Numerous systems have been developed for the overexpression of recombinant proteins and can be adapted for antibody fragment production. The method described in this chapter relies on the lactose promoter–repressor–operator system. Other systems may work equally well, and the methods given can be adapted accordingly. While moderate amounts of most, if not all, Fabs can be expressed with these methods, high-level expression may require engineering of the antibody fragment to maximize expressibility in E. coli (see Note 1). In the following protocols, the expression of a humanized Fab' fragment is used to exemplify the methodology. A description of small-scale production by isopropyl-β-D-thiogalactopyranoside (IPTG) induction in shake flasks
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ورودعنوان ژورنال:
- Methods in molecular biology
دوره 308 شماره
صفحات -
تاریخ انتشار 2005