The synthesis of Fab was performed by coexpression of immunoglobulin L- and H-chains using the method and components previously described, henceforth called the disulfide insect system. For the expression of anti-lysozyme Fab, two plasmids (pIX5.0-Mel-LaLys and pIX5.0-Mel-HaLys) encoding L- and H-chains were constructed via conventional cloning. Two linear templates, Mel-VLCL-SII (L-chain) and Mel-VHCH1 (H-chain), were produced for the synthesis of anti-CD4 Fab via expression PCR. In contrast to procedures described earlier, we provided the L- and H-chains with a signal peptide in order to direct the synthesis into microsomal vesicles.
To determine whether Fab antibody fragments are produced as dimers of L- and H-chains, anti-lysozyme Fab and anti-CD4 Fab were expressed using the disulfide insect system as described previously. After cell-free coexpression, reaction samples were separated by reducing and non-reducing 15% SDS-PAGE, the latter to maintain the integrity of the intermolecular disulfide bonds. The radiolabeled immunoglobulin chains were detected by autoradiography. Whereas the chains expressed in separate synthesis reactions do not form self dimers (data not shown), the co-expressed L- and H-chains are detected as dimers with increased apparent molecular weight in the non-reducing part of the gel (Figure 1). The conversion from monomeric to dimeric Fab chains is almost complete.
As the proteins that are synthesized in the disulfide insect system are expected to be translocated into microsomal vesicles by means of the melittin signal peptide, a direct interaction of these proteins with their target molecules is not possible without disruption of the vesicular lipid membrane. Therefore, we examined how a protein that has been translocated into vesicles can be made accessible to an application. Since N-glycosylation is seen as a proof for previous translocation, we used the glycoprotein Erythropoietin (Epo) as a model to analyze the translocation into the vesicles and the subsequent release of synthesized proteins by treatment with detergent.
A radioactively labeled Epo mutant containing the melittin signal peptide and one N-glycosylation site was expressed using the plasmid pIX2.0-Mel-Epo-N52+110Q as described in Materials and methods. Glycosylation was verified by enzymatic deglycosylation of reaction samples as described (27). After Epo synthesis, reactions were treated with different concentrations of detergent and subsequently centrifuged at 16.000× gfor 10 min. Under these conditions the vesicles including trapped Epo were pelleted, whereas after successful lysis the protein should remain in the supernatant. Indeed, at a concentration of 0.05% Brij 35 the vesicles are completely lyzed as indicated by the complete release of Epo and its recovery in the supernatant. Previous translocation into microsomal vesicles is indicated by the N-glycosylation (Figure 2).
Next the biological activity of Fab synthesized with the disulfide insect system was analyzed by the ability of anti-lysozyme Fab to inhibit the lytic activity of lysozyme. As a control, co-expression was performed in a commercially available standard system that also contains active microsomal vesicles but exhibits reducing conditions (EasyXpress Insect Kit II, Qiagen). As expected, activity of anti-lysozyme Fab can only be detected, if Fab is synthesized using the disulfide insect system containing oxidized and reduced glutathione, whereas there is no activity resulting from synthesis using the standard system (Figure 3A).
Since we provided the Fab chains with signal peptides for translocation into vesicles these results show that active Fab can be produced in cell-free systems using the combination of redox conditions and the natural translocation mechanism, and that translocation into microsomal vesicles might be useful for the synthesis of active Fab in such systems.
In order to examine the contribution of translocation to Fab activity, we removed the microsomes mostly from the disulfide insect system by centrifugation prior to protein synthesis. Overall synthesis yield was measured via incorporation of radioactively labeled amino acids as described in Materials and methods, and the specific activities of anti-lysozyme Fab synthesized in the presence and absence of microsomes were analyzed. For that purpose, the inhibiting effect of Fab on lysozyme activity was measured. Fab synthesized in the presence of microsomes shows activity, which is approximately one order of magnitude higher compared with Fab synthesized without microsomes (Figure 3B).