Expression of Antibody Fragments by Periplasmic Secretion in Escherichia coli

Andrew G. Popplewell, Mukesh Sehdev, Mariangela Spitali, and A. Neil C. Weir

1. Introduction

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 expres-sibility 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-P-d-thiogalactopyranoside (IPTG) induction in shake flasks

From: Methods in Molecular Biology, vol. 308: Therapeutic Proteins: Methods and Protocols Edited by: C. M. Smales and D. C. James © Humana Press Inc., Totowa, NJ

is followed by a protocol for fermentation using a switch of carbon source from glycerol to lactose as the means of induction. Methods for periplasmic extraction and purification are also given.

2. Materials

1. Plasmids: pTTO-1 and pDNAbEng-1 (Celltech R&D).

2. E. coli strains: INVaF' (InVitrogen) and W3110 (ATCC).

3. Oligonucleotide primers.

4. Restriction enzymes, T4 DNA ligase and Taq DNA polymerase.

5. Agarose gel apparatus and DNA sequencing apparatus.

6. L-broth: 10 g/L bactotryptone, 10 g/L NaCl, 5 g/L yeast extract. Sterilize by autoclaving.

7. Transformation solution: 10 g/L bactotryptone, 10 g/L NaCl, 5 g/L yeast extract, 10 g/L MgCl2, 100 g/L polyethylene glycol (PEG)4000, and 50 mL/L dimethylsulfoxide (DMSO), pH 6.5. Filter-sterilize.

8. SOC medium: 2% bactotryptone, 0.5% yeast extract, 10 mMNaCl, 2.5 mMKCl, 10 mMMgCl2, 10 mM MgSO4, and 20 mM glucose (added from 20% stock solution after autoclaving other components).

9. Tetracycline: 7.5 mg/mL stock solution in 50% ethanol.

10. Glycerol solutions: 80% (w/w), 52.5% (w/w). Sterilize by autoclaving.

11. IPTG: 200 mM stock solution, make fresh. Filter-sterilize.

12. Periplasmic extraction buffer (2X stock): 200 mM Tris-HCl and 20 mM EDTA, pH 7.4.

13. Coating buffer: 1.59 g/L Na2CO3, 2.93 g/L NaHCO3, and 0.2 g/L NaN3, pH 9.6.

14. Capture and reveal antibodies.

15. Phosphate-buffered saline (PBS).

16. PBS-Tween (PBST): PBS plus 0.05% (v/v) Tween-20.

17. Sample conjugate buffer: 0.1 M Tris-HCl, 0.1 M NaCl, 0.02% (v/v) Tween-20, and 0.2% (w/v) casein, pH 7.0.

18. Substrate solution: 10 mL sodium acetate/citrate solution (0.1 M, pH 6), 100 |JL H2O2 solution (0.44% [v/v]), and 100 |lL tetramethyl benzidine solution (10 mg/mL in DMSO).

19. 2.5 L Braun BiostatB batch fermenter (or equivalent) and associated pH and dissolved oxygen probes.

20. SM6 defined media: 5.2 g/L (NH4)2SO4, 4.2 g/L NaH2PO4-2H2O, 4.025 g/L KCl, 1.05 g/L MgSO4-7H2O, 5.2 g/L citric acid, 0.052 g/L CaCl2-2H2O, 0.0200 g/L ZnSO4-7H2O, 0.0275 g/L MnSO4-4H2O, 0.0075 g/L CuSO4-5H2O, 0.004 g/L CoSO4-7H2O, 0.1000 g/L FeCl3-6H2O, 0.0003 g/L H3BO3, 0.0003 g/L Na2MoO4-2H2O, and 31.1 g/L glycerol. Sterilize by autoclaving.

21. Struktol (antifoam agent) stock solution: 10% (v/v). Sterilize by autoclaving.

22. Lactose solution: 40% (w/w). Sterilize by autoclaving.

23. Sartobrand P capsule (Sartorious) fitted onto a peristaltic pump and tubing.

24. Nalgene stericups: 0.45 |lm and 0.22 |lm (Millipore).

25. Affinity chromatography equilibration buffer: PBS.

26. Affinity chromatography elution buffer: 0.1 M glycine-HCl, pH 2.7.

27. Protein G Gammabind Plus Sepharose (Amersham Biosciences).

3. Methods

These methods outline the (1) construction of the expression plasmid; (2) small-scale expression of the protein in E. coli; (3) large-scale expression by fermentation; and (4) extraction and purification of the protein.

3.1. Construction of the Expression Plasmid

The construction of a plasmid for expression of a Fab' fragment by translocation to the E. coli periplasm is described. The same principle can be applied to construct plasmids to express other antibody fragments, including scFv. However, the construction is more complicated for those fragments made up of two component polypeptides, such as the Fab'. Such "double gene" expression can be achieved by the use of separate promoters for each gene, but perhaps the simplest solution is to use a dicistronic expression method as described here. This indicates that two genes are produced from one transcript, requiring only a single promoter.

3.1.1. pTTO Expression Vector

Plasmid pTTO-1 (Fig. 1) is a derivative of plasmids pTTQ9 (3) and pACYC184 (4). The expression unit consists of the strong Tac promoter (5) and dual rrnBt1t2 transcriptional terminator (6). The plasmid contains the laclq gene (7), giving constitutive expression of the lac repressor protein necessary to keep the tac promoter repressed. Derepression (or induction) is mediated by the addition of lactose (which is converted to allolactose, the natural inducer of the lac operon in E. coli), or IPTG, a non-hydrolyzable synthetic inducer. The plasmid also contains the origin of replication from plasmid p15A (8), conferring a low-copy number, and it carries the tetracycline resistance gene. As shown in Fig. 1, the plasmid contains DNA encoding a portion of the signal peptide from the E. coli OmpA protein (9). Insertion of DNA encoding an antibody fragment and the remainder of the OmpA signal sequence creates a gene encoding a protein product that will be translocated to the E. coli periplasm.

3.1.2. Insertion of Fab' Light-Chain Gene

The light chain of a Fab' can be inserted into the pTTO-1 polylinker so that an in-frame OmpA signal peptide is created on ligation. A polymerase chain reaction (PCR) strategy is required to "build" DNA encoding the C-terminal portion of the signal peptide onto the 5' end of the light-chain gene. A suggested oligonucleotide to act as a 5' forward PCR primer is shown in Fig. 1. Thus, the unique Mfel site within the pTTO-1 polylinker sequence is employed. For the 3' reverse primer, no additional components are necessary, apart from a restriction site for cloning into the vector BsiWI site. The cloning scheme is as follows (see Note 2):

1. Use PCR to amplify the light chain, adding a 5' MfeI site and DNA encoding part of the OmpA signal peptide and adding a BsiWI site to the 3' end of the light-chain gene.

2. Purify the amplified DNA, digest with restriction enzymes MfeI and BsiWI, and repurify.

3. Prepare vector pTTO-1 by restriction with MfeI and BsiWI, and purify the cleaved fragment.

4. Ligate the two fragments together, and transform into an appropriate host strain (see Note 3).

This creates the light-chain intermediate plasmid. This method can be adapted for creation of a plasmid for single-gene expression (e.g., scFv), in which case the expression construct is now complete. For Fab' expression, the heavy-chain gene must also be inserted.

Plasmid Digestion Procedure

Fig. 1. Expression plasmid pTTO-1. (A) Map of plasmid. P Tac, tac promoter and lac operator region; SP, signal peptide; rrnBt1t2, transcriptional terminator; lacIq, gene constitu-tively expressing lac repressor protein; p15A ori, origin of replication; Tet R, tetracycline resistance gene. (B) Nucleotide sequence of polylinker showing N-terminal portion of OmpA signal peptide amino acid sequence. Restriction enzyme recognition sequences are underlined. The ribosome binding site (RBS) is shown in bold. (C) Potential sequence of oligonucleotide to add remainder of OmpA signal peptide sequence to sequence of antibody fragment gene. N, any nucleotide; X, nucleotide identical to that in antibody fragment to be amplified.

Fig. 1. Expression plasmid pTTO-1. (A) Map of plasmid. P Tac, tac promoter and lac operator region; SP, signal peptide; rrnBt1t2, transcriptional terminator; lacIq, gene constitu-tively expressing lac repressor protein; p15A ori, origin of replication; Tet R, tetracycline resistance gene. (B) Nucleotide sequence of polylinker showing N-terminal portion of OmpA signal peptide amino acid sequence. Restriction enzyme recognition sequences are underlined. The ribosome binding site (RBS) is shown in bold. (C) Potential sequence of oligonucleotide to add remainder of OmpA signal peptide sequence to sequence of antibody fragment gene. N, any nucleotide; X, nucleotide identical to that in antibody fragment to be amplified.

3.1.3. Insertion of the Fab' Heavy-Chain Gene

To create a dicistronic message expressing both Fab' chains, the gene for the heavy chain also needs to be inserted 3' to the light-chain gene. Another signal sequence is required, in addition to a ribosome-binding site to ensure efficient translational initia-

A Restriction Map of Plasmid

Bglll EcoRV Clal EcoRI Xbal JVIfel

Bglll EcoRV Clal EcoRI Xbal JVIfel

Fab Expression Plasmid

B Sequence of Polylinker 1

Bgl 11 EcoRV Clal JicoRI Xbal RBS Mfel

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