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Chapter 21 : Periplasmic Expression of Antibody Fragments

Author: David P. Humphreys1
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Affiliations: 1: UCB-Celltech, Slough SL1 4EN, United Kingdom
Content Type: Monograph
Publication Year: 2007

Category: Bacterial Pathogenesis; Microbial Genetics and Molecular Biology

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Abstract:

This chapter focuses on the biology and practical techniques that can help in the expression of antibody fragments in . The periplasm is often preferred for expression of soluble antibody fragments and some of the factors for this choice are discussed. The periplasm is the site of expression for only ~1/10 of the soluble proteins of and does not contain any DNA or RNA that can interfere with purification. Therefore combination of periplasmic expression with carefully considered harvest and extraction regimes can result in a very useful concentration and partial purification/enrichment of the periplasmic protein of interest. An important factor for enabling useful levels of protein expression is good selection or design of the expression plasmid. The key areas considered here are: (i) plasmid copy number/ origin of replication, (ii) constitutive versus inducible promoter, (iii) resistance/selection marker, and (iv) design of the coding region. The promoter along with depletion of phosphate has also been used to good effect for the production of antibody fragments. A notional advantage of periplasmic expression over extracellular expression is the ability to concentrate the product simply by harvesting the cells by centrifugation or filtration and avoiding vortex-type protein damage. The chapter describes various designs of antibody fragments along with the engineered adaptations and improvements to both the fragment and the host. The endoplasmic reticulum of eukaryotes is the natural site for the folding and assembly of antibodies.

Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21

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Periplasmic Expression of Antibody Fragments
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Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21
/content/10.1128/9781555815806.ch21.ch21fig01
FIGURE 1
Schematic representation of full-length IgG1 (human 71 isotype). Native IgG consists of two heavy-chain and two light-chain polypeptides. Each heavy chain has four domains: one variable (Vh) and three constant (C1-hinge-C2-C3). Each light chain has two domains: one variable (V) and one constant (C). The two heavy chains are covalently linked by disulfide bonds between hinges and each light chain is attached to a heavy chain by a disulfide bond. In native human IgG carbohydrate is attached to each heavy chain at Asn297 of the C2 domains.
10.1128/9781555815806/f0363-01_thmb.gif
10.1128/9781555815806/f0363-01.gif
Image of FIGURE 1
FIGURE 1

Schematic representation of full-length IgG1 (human 71 isotype). Native IgG consists of two heavy-chain and two light-chain polypeptides. Each heavy chain has four domains: one variable (Vh) and three constant (C1-hinge-C2-C3). Each light chain has two domains: one variable (V) and one constant (C). The two heavy chains are covalently linked by disulfide bonds between hinges and each light chain is attached to a heavy chain by a disulfide bond. In native human IgG carbohydrate is attached to each heavy chain at Asn297 of the C2 domains.

Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21
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Tables

Periplasmic Expression of Antibody Fragments
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Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21
/content/10.1128/9781555815806.ch21.ch21tab01
TABLE 1
Antibody fragments produced in
Generic image for table
TABLE 1

Antibody fragments produced in

Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21
/content/10.1128/9781555815806.ch21.ch21tab02
TABLE 2
Protein sequences of useful signal peptides
Generic image for table
TABLE 2

Protein sequences of useful signal peptides

Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21
/content/10.1128/9781555815806.ch21.ch21tab03
TABLE 3
Examples of translational fusion partners for periplasmic proteins
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TABLE 3

Examples of translational fusion partners for periplasmic proteins

Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21
/content/10.1128/9781555815806.ch21.ch21tab04
TABLE 4
Examples of fusion partners and cleavage regimes for periplasmic expression of antibody fragments
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TABLE 4

Examples of fusion partners and cleavage regimes for periplasmic expression of antibody fragments

Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21
/content/10.1128/9781555815806.ch21.ch21tab05
TABLE 5
Comparison of periplasmic extraction methods
Generic image for table
TABLE 5

Comparison of periplasmic extraction methods

Citation: Humphreys D. 2007. Periplasmic Expression of Antibody Fragments, p 361-388. In Ehrmann M (ed), The Periplasm. ASM Press, Washington, DC. doi: 10.1128/9781555815806.ch21

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