Directed evolution of single-chain Fv for cytoplasmic expression using the β-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation
© Philibert and Martineau; licensee BioMed Central Ltd. 2004
Received: 08 December 2004
Accepted: 17 December 2004
Published: 17 December 2004
Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli.
We used the β-galactosidase α-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of β-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the β-galactosidase activity present in the cells.
This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free α-peptide released from the protein fusion by the host proteases. This means that the α-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released α-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein.
Because of their high affinity and specificity against their antigen, antibody molecules and their fragments have many applications in research, diagnosis and therapy . E. coli is a widely used organism for the production of proteins, including antibody fragments such as Fab or single chain variable domains (scFv). Active scFv can be obtained by targeting the protein to E. coli periplasm, where the two disulfide bonds needed for protein folding and stability [2–4] can form. The amount of scFv produced is however usually low, in the range of 0.1–1 mg l-1 of culture at an optical density (OD600) of 1, even if expression levels of 10 mg l-1 have sometimes been reported .
An alternative strategy to produce antibody fragments in E. coli has been to maintain the scFv in the cytoplasm by removing its signal sequence. Under those conditions, scFv might be expressed at very high levels, albeit in an insoluble and inactive conformation. Even if highly efficient in vitro refolding procedures have been developed for scFv and Fab [6, 7], it would be more suitable to directly recover soluble active protein from the cytoplasm of the cell. This has been partially accomplished by modifying the cytoplasmic oxido-redox environment by mutating components of the thioredoxin and glutaredoxin pathways , resulting in the accumulation of soluble intra-cytoplasmic oxidized antibody fragments [9–11]. The soluble expression levels reported, however, are not higher than those obtained in the periplasm of the cell. Another interest in expressing soluble active scFv in the reducing environment of the cytoplasm is that the expression of scFv molecules inside the cell can be used to block viral replication and to inhibit oncogene products [12–15]. The use of so-called intrabodies opens many interesting possibilities in gene therapy  and in the in vivo study of protein function .
Several methods have been proposed in recent years to increase the soluble expression levels of foreign proteins expressed in E. coli cytoplasm . Most of these methods rely on fusion between the protein of interest and a reporter enzyme. If the protein folds into a soluble conformation, the fused enzyme will be active; but if the protein ends up as an inclusion body, the fused enzyme will be inactive, resulting in a null phenotype. Three reporter enzymes have been used to date, the green fluorescent protein (GFP ), the chloramphenicol acetyl transferase (CAT ) and the β-galactosidase (βgal ). In the case of βgal, the whole enzyme was not fused to the protein of interest but only a small N-terminal fragment of about 50 aminoacids called the α-fragment . The inactive enzyme remainder was expressed in trans by the bacteria (β-galactosidase ΔM15 protein), resulting in in vivo complementation and a lactose+ (lac+) phenotype . The role of the α-fragment is to promote the tetramerization of the inactive dimeric ΔM15 mutant , resulting in βgal activation.
Using the GFP fusion method, Waldo and collaborators [18, 25, 26] isolated several soluble variants of aggregating proteins. However, since the method is based on a phenotypic screen, it is limited to the exploration of libraries of about 105 clones and does not allow the isolation of mutants from very large libraries. This is not the case for the two other methods which should allow the selection of very rare events by selecting for chloramphenicol resistance or growth on lactose as the carbon source. To date, there is, however, no report of protein evolution and selection using these latter two methods even if the CAT system has been used to pre-select libraries of hybrid proteins to avoid stop codons and enrich in properly folded molecules .
We previously showed that it is possible to evolve an scFv molecule for very high expression levels in E. coli cytoplasm [4, 28, 29]. This evolved scFv is active in the cell and its expression level is as high as 100 mg l-1 at an OD600 of 1. This was, however, a very rare event, and the selection had to be conducted using large libraries of 107–108 mutants. The selection procedure used was restricted to the properties of a particular antibody molecule able to activate βgal mutants in vitro and in vivo. To extend this result to other scFv molecules, we constructed fusions between two scFv and the α-fragment of βgal to monitor the soluble expression level in vivo. The lactose phenotype of the strain was then used to select mutants with improved lactose utilization and thus presumably expressing scFv at a higher level in the bacterial cytoplasm.
The level of α-complementation was further characterized by measuring the βgal activity present in the cell using the whole cell assay developed by Miller . The result shown in Fig. 2B demonstrated that the βgal activity in TG1(pPM173) was close to background level and that the activity increased gradually to 100% of the activity of the positive α-complementation control. This clearly demonstrated the correlation between the soluble cytoplasmic expression levels of the scFv and the lactose phenotype of the strain, opening the way to direct selection of mutant proteins using this assay.
Spontaneous lactose plus mutants
When TG1(pPM173) was incubated 2 to 3 days at 37°C, red papillae appeared on the MacConkey plates. These clones were isolated and showed a stable lac+ phenotype, comparable to a wild type lac+ strain. One of the clones, 173S1, had even a higher βgal activity in its cytoplasm than the pUC119 rescued ΔM15 strain (Fig. 2B). The increase in βgal activity was shown to be linked to the plasmid by transforming TG1 with the plasmid extracted from TG1(pPM173S1). To determine if the mutation was located in the scFv, we excised the gene from the plasmid using the Nco I and Not I sites and cloned it back in pPM170. The resulting clone was lac- (data not shown), demonstrating that the mutation responsible for the lac+ phenotype was associated with the plasmid but not with the scFv.
To understand the origin of this phenotypic change, we sequenced the α-fragment of two of the clones, 173S1 and 173S2. Both contained a mutation in the α-fragment of the βgal, resulting in a stop codon (Fig. 1) at positions 49 and 50. It should be noted that the stop codon of 173S1 is an amber codon which is partially suppressed in TG1 and replaced by a Gln, resulting in this case in two different fusions, one stopping at position 49 of the βgal and the second with a complete α-fragment, showing that the mutant is dominant over the wild type.
Analysis of the sequence of the α-fragment present in pUC119 (and pPM170), showed that a 30 aminoacid long peptide of unknown origin was fused to the C-terminal extremity of the α-fragment (Fig. 1). This peptide (or part of it) was present in all the pUC derived plasmids. Sequence analysis showed that this peptide originated from a fusion between a DNA sequence present in pBR322 plasmid between the Nde I and Ear I sites (nucleotides 2297 to 2351 of pBR322; LMR...HRI translated sequence) and the 3' end of M13 gene IV (nucleotides 5470 to 5511 of M13; RQS...AAH translated sequence). The pBR322 sequence, located between the rop gene and the origin of replication, is not normally translated, and the M13 gene IV sequence is read out of frame.
However, despite a strong increase in the expression level of 173S1-R4 versus 173S1, the lactose phenotypes of both clones expressed in strain TG1 were comparable (Fig. 3A). This showed that the link between the lactose phenotype and the intracellular expression levels of scFv13 shown in Figure 2 was due to the presence of the 30 aminoacid long peptide. Indeed, in the case of 173S1, the α-complementation detection system is saturated and the strong lactose phenotype of strain TG1(173S1) would not allow the selection of evolved mutants with a stronger phenotype. This prompted us to retain this foreign 30 aminoacid long peptide for selection and to adapt the procedure to avoid the selection of spontaneous stop codons.
Two scFv were chosen as models. We first used a humanized D1.3 anti-lysozyme antibody, HuLys11 . This antibody has been extensively studied and its structure solved by X-ray crystallography. The second scFv was derived from the mouse monoclonal antibody 225.28S and recognizes the high molecular weight melanoma-associated antigen . As such, this antibody might have potential applications in the treatment of that type of cancer.
To confirm the phenotype shown on the plates, βgal activity was measured using the assay described by Miller . The assay relies on cell lysis by chloroform/SDS and as such measures the amount of βgal activity present in the whole cell. As shown in Figure 5B, the βgal activity present in the mutants was about five to seven times higher than in the strain expressing the wild type 225.28S scFv fused to the α-fragment and five times lower than in the control cells expressing the unfused α-fragment from the pUC119 plasmid. In oder to confirm that this increase in βgal activity was localized in the soluble protein fraction, we measured the activity in soluble extracts of the cells prepared by lysozyme treatment and centrifugation. The results were comparable to those obtained with the whole cell assay (Fig. 5B), demonstrating that indeed the increase in βgal activity was due to an increase in α-complementation in the soluble protein fraction.
Fused scFv-α characterization
To test this hypothesis, we expressed the clones in a TG1 strain deficient in the cytoplasmic Lon protease. This protease has been shown to be involved in the degradation of many recombinant proteins in E. coli . When expressed in such a strain, the wild type scFv225.28S was detected in the soluble fraction, albeit at a very low level (Fig. 7C), and no scFv was present in the insoluble fraction. All the evolved scFv were expressed at a much higher level than the wild type, mainly as soluble proteins (Fig. 7C &7D), showing that the scFv225.28S proteins were predominantly degraded by the Lon protease in vivo. The same type of result was obtained with scFv HuLys11 since the wild type scFv was not detected in any of the extracts, and the evolved scFv were detected in the soluble fraction of the Lon deficient strain (Fig. 7F&7G). The Lon protease is not the only protease present in the E. coli cytoplasm  and in its absence some degradation of the fusion is still present as shown by the additional band at about 30 kDa in Fig. 7C &7D. This molecular weight is consistent with a degradation site located between the scFv and the α-peptide and could be one of the sources of the free α-peptide liberated in the cell cytoplasm (see below). It should be noted that in the reported cases of the successful isolation of soluble variants of aggregating proteins using the GFP system [25, 26], the authors used an E. coli B strain, naturally deficient in the Lon protease [33, 35].
The soluble extracts of scFv225.28S and its mutants were tested for the presence of βgal activity (Fig. 7E). The results obtained in strain TG1 were comparable, as expected, to those obtained previously (Fig. 5). There is however no clear correlation between the soluble expression level and the βgal activity since clone R4.1, which is expressed at the highest level, did not give a higher signal than the non-detected clones, R4.2 and R4.4. In the Lon-deficient strain, all the clones, including the wild type scFv225.28S, gave the same signal despite the fact that the wild type scFv was only barely detectable in the extract. These results showed that there was no correlation between the soluble expression level of the fusion protein and the βgal signal. The most likely explanation of this phenomenon is that the βgal signal is not due to the fusion protein detected by western blot but to some α-peptide released from the fusion by the host proteases. This free α-peptide is presumably also much more efficient for βgal complementation than the fusion protein since it may penetrate the tunnel present in M15 protein  at a much faster rate.
Unfused scFv characterization
We next compared the expression level of the best isolated clone (scFv225.28S R4.1) in the periplasm and in the cytoplasm. It must be noted that the two vectors used, pPM210 and pAB1, are derived from the same pUC119 plasmid and that in both cases the scFv gene is under the control of the lac promoter. The scFv expressed by the two plasmids are exactly the same except for the pel B leader peptide present at the N-terminal extremity of the scFv expressed in the periplasm. After cleavage of this signal sequence, the two scFv only differ by an additional N-terminal Met-Ala dipeptide present in the scFv produced in the cytoplasm.
In this report, we used the α-complementation assay as a probe to detect soluble scFv expression in E. coli cytoplasm . The system was used to evolve two scFv to increase their expression levels. After four rounds of mutation and selection, we were able to select for scFv fusions giving high βgal activity in vivo and in vitro.
Characterization of these mutant proteins showed that they were expressed at higher levels in the cytoplasm than the wild type scFv. The proteins were however quickly degraded in the cell cytoplasm, and only a faint band was detected in the soluble fraction and no protein at all in the insoluble fraction (Fig. 7). This is not surprising for scFv expressed in the reducing environment of the cytoplasm since the lack of the two disulfide bonds results in only marginally stable proteins quickly degraded by the host proteases . Since it has been shown that the Lon protease is involved in the degradation of many recombinant proteins in E. coli , we expressed the scFv in an isogenic strain deficient in this protease. Under those conditions, the mutant scFv were expressed at a much higher level than the wild type scFv. This was true for both scFv, HuLys11 and 225.28S.
This increase in soluble cytoplamic production of the scFv-α fusion was however not correlated with the βgal activity (Fig. 7). For example, clone scFv225.28S R4.1 and R4.2, which were expressed at a very different soluble level (Fig. 7C), gave the same βgal signal (Fig. 5 &7E). This indicates that most, if not all, of the βgal activity present in the cell was not due to the scFv-α-peptide fusion but to some free α-peptide released from the fusion by the host proteases. This means that the α-complementation assay does not sense the scFv-α expression level, as hypothesized, but rather the amount of free α-peptide in the cell cytoplasm. This could also explain why the system was not able to detect differences between the clones in a lon background (Fig. 7E).
There is a striking difference between the properties of the fused and the unfused scFv. Indeed, in the structural context of the selection (as fusion to the α-peptide), all the scFv, albeit highly sensitive to degradation, were mainly expressed as soluble proteins. This is particularly the case of HuLys11 for which no insoluble protein was detected in Fig. 7G. As unfused scFv, all the clones were, however, mainly found in the insoluble fraction, particularly when expressed using the strong T7 promoter (Fig. 10) but also when we used the same promoter than during the selection process (Fig. 8). This shows that interactions could take place between the scFv and the fused α-peptide either during or after folding of the fusion protein. This may explain why all the mutations where localized on the surface of HuLys11 (Fig. 6) since these residues are more likely to take part in such an interaction. This is particularly the case of the H46 mutation, which is present in all three isolated mutants. In this case a hydrophilic and charged residue (Glu) is replaced by an hydrophobic Gly. As the α-peptide and the 30 aminoacid peptide fused to the scFv contain some highly hydrophobic patches of residues (data not shown), this increase in the hydrophobicity of the scFv surface may favor interaction between the two partners that could enhance the solubility of the fusion by a chaperone-like effect, as proposed for other fusions . When the scFv is expressed alone, without the C-terminal α peptide, increasing the hydrophobicity of the surface residues could result in an increase in aggregation , as noted in our case (Fig. 8 &10).
Finally, another problem during the selection originated in the presence of the 30 aminoacid peptide at the end of the α-peptide. As we showed in the case of the model antibody scFv13, this peptide is needed to obtain good correlation between protein soluble expression levels and in vivo α-complementation since the introduction of a stop codon at the end of the α-peptide resulted in a very strong complementation with all the scFv, even the wild type (clone 173S1 in Fig. 2 &3). This may explain why the selection was biased towards degraded C-terminal α-peptide. Indeed, Figure 3 shows that the role of the additional 30 aminoacid long C-terminal peptide is to decrease the expression level of the fusion in order not to saturate the detection system with the wild type scFv. This decrease in the expression level is presumably accomplished through targeting of the fusion to the cell degradation systems, resulting in a rapid conversion of the fusion to free α-peptide and in a bias in the selection procedure towards degradation and thus aggregation. It must be noted that Schwalbach and collaborators  recently selected a mutant scFv using the GFP system, and that despite an increase in GFP activity, they did not notice any increase in soluble protein expression but rather an increase in protein aggregation. They also noticed that protease degradation of the fusion released free GFP in the cytoplasm, particularly when cells were induced for long periods, that is, the conditions used during the selection process. It should be noted that the authors used a lon+ strain for the selection instead of the E. coli B lon- strain used by other authors [25, 26, 35]. These failures may be due, in the specific case of cytoplasmic scFv expression, to the lack of disulfide bond formation, leading to marginally stable reduced scFv, quickly degraded by the cell proteases .
Despite the difficulties associated with cytoplasmic scFv expression, some scFv have been previously evolved for folding under reducing conditions [28, 51]. The systems used relied on the binding activity of the scFv molecule, avoiding the problem of protein degradation since degraded antibody cannot bind to its antigen anymore. It would thus be more appropriate to use yeast or bacterial two-hybrid systems to evolve scFv [52, 53]. The use of such systems could also avoid the selection of mutations that modify or abolish the antigen-binding properties of the scFv as is presumably the case in our selection since several of the mutations are located in the CDR loops of the antibody fragments (see additional files 4 and 2, HuLys11aa.txt and 22528Saa.txt). Another possibility could be to use E. coli protease-deficient strains to limit protein degradation during the selection, but the results shown in Fig. 7E demonstrate that even in such a strain there is no correlation between the soluble expression level and the βgal activity present in the cell.
In this report, we used the α-complementation assay as a probe to detect soluble scFv expression in E. coli cytoplasm. The system was used to evolve two scFv in order to increase their expression levels. After four rounds of mutation and selection, we were able to select for scFv fusion giving a high βgal activity in vivo and in vitro.
Characterization of these mutant proteins showed that their expression levels were much higher in the cytoplasm than those of the wild type scFv, particularly in a Lon-deficient strain. There was however no correlation between the βgal activity present in the cell and the soluble expression level of the scFv, showing that the βgal signal is presumably due to some free α-peptide released from the fusion protein by the host proteases and not to the non-degraded soluble scFv-α fusion.
Media, plasmids and bacterial strains
MacConkey agar, M9 and LB media were previously described . Strain TG1 is E. coli K-12, [F' traD 36 lac Iq Δ(lac Z)M15 pro A+B+] sup E44 Δ(hsd M-mcr B)5 thi Δ(lac-pro AB). CAG626F' is E. coli K-12, [F' lac IqΔ(lac Z)M 15 zzf::mini-Tn 10(KanR)pro A+B+] lac Z(am) pho(am) lon trp(am) tyrT [supC(ts)] rpsL mal(am) .
TG1lon strain was constructed by P1 transduction. First, zaj-3054::Tn 10, located at 9.95 minutes, was inserted near to the lon gene in strain CAG626F' by P1 transduction from CAG12017 . In a second step, lon and zaj-3054::Tn 10 were co-transduced in TG1 using P1 phage. The introduction of the lon mutation was verified by comparing the resistance to UV irradiation and to nitrofurantoin of TG1 and TG1lon.
Plasmid pPM170 for the cytoplasmic expression of scFv fused to the α-fragment of βgal was constructed as follows (Fig. 1). A fragment containing a Shine Dalgarno sequence followed by a Nco I site containing the ATG initiator was obtained by PCR amplification of pPM160  with pTrpFOR (CGGGAATAAGCTTCAACGCCAG) and EcoAlpha.for (GTGAATGAATTC GAATGGTGATGATGG) primers. The underlined sequence corresponds to an Eco RI site designed in order to get in frame fusion of the scFv with the α-fragment of βgal present in pUC119 when the fragment was cloned in the Eco RI site of pUC119. The amplified fragment also contained an Hpa I site located 34 bp upstream from the ATG initiator of the scFv gene. This PCR band was digested with Hpa I and EcoR I enzymes and ligated with Hinc II-Eco RI-digested pUC119 plasmid, leading to the pPM170 plasmid.
Plasmid pAB1 for the periplasmic expression of scFv under the lac promoter has been described previously . Plasmid pPM210 is identical to pPM170 except for the presence of a stop codon between the scFv and the α-fragment of βgal. It was obtained by cloning the Nco I-Eco RI fragment of pAB1 in the Nco I and Eco RI sites of pPM170. scFv cloned in pPM210 were transcribed from a lac promoter and tagged at their C-terminal extremity by both a c-myc and a polyhistidine flags.
Plasmid pET23NN was designed to easily clone Nco I-Not I fragment containing scFv from pPM170-derived plasmids under the T7 promoter with C-terminal c-myc and His6 tags. pET23d(+) obtained from Novagen was first digested with Xho I enzyme, filled-in with T4 DNA polymerase then digested with Nco I. This fragment was ligated with a Nco I-Eco RI(filled) fragment excised from the pAB1 plasmid . The resulting plasmid contained a T7 promoter, followed by a Nco I site containing the ATG initiator, a Not I site followed by a c-myc and a His6 tag [same sequence as pPM170 (Fig. 1) but with a stop codon before the Eco RI site]. Due to the removal of a T, presumably during T4 DNA polymerase treatment, ligation between the filled EcoR I and Xho I sites reconstituted the EcoR I site (GAATTCGAG instead of GAATTTCGAG).
The conditions used are those described to obtain 0.2% mutations with p(AT->NN) = p(GC->NN) and p(AT->GC) = p(AT->TA) for ten duplications [28, 56]. The amplified band was digested with Nco I and Not I and cloned in pPM170. The ligation was transformed in TG1 by electroporation. Bacteria were plated on MacConkey lactose and M9 lactose agar plates supplemented with 100 μg ml-1 ampicillin, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 1 μg ml-1 of vitamin B1 in the case of the M9 plates.
Preparation of bacterial extracts
Expression under the T7 promoter was conducted essentially as described except that LB was used instead of M9ZB . Freshly transformed cells were grown in LB supplemented with 100 μg ml-1 ampicillin and 20 μg ml-1 chloramphenicol until an OD600 of about 1 and kept at 4°C overnight. In the morning, the cells were diluted to an OD600 of 0.1 in the same medium and incubated with shaking for 2 hours at 37°C, then 0.4 mM IPTG was added and the culture continued for 3 hours at 30°C. The cultures were cooled down on ice for 5 to 30 minutes, then centrifuged and resuspended at a concentration of 40 OD600 in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Cells were lysed by freezing/thawing followed by a brief sonication. The insoluble fraction was collected by centrifugation (30 minutes at 17 500 g), washed once, then resuspended in the same volume of buffer.
For expression under the lactose promoter (plasmid pPM170), an overnight culture in LB supplemented with 100 μg ml-1 ampicillin and 1% glucose was diluted to a final OD600 of 0.1 in LB supplemented with 100 μg ml-1 ampicillin and incubated with shaking for 2 hours at 37°C, then 1 mM IPTG was added and the culture continued for 3 hours at 30°C. Cytoplasmic soluble extracts were prepared as follows. 16 OD of culture were cooled down on ice for 5 to 30 minutes, then centrifuged and resuspended in 400 μl of 20 mM Tris-HCl (pH 8.0), 0.7 M sucrose. Hen egg-white lysozyme at a 0.1 mg ml-1 concentration was added and the extract incubated 2 minutes on ice. 800 μl of a cold solution of 1.5 mM EDTA were added slowly within ten minutes. Extract were kept 30 minutes on ice then sonicated 20 s. The insoluble fraction was collected by centrifugation (30 minutes at 17 500 g), washed once, then resuspended in the same volume of buffer than the soluble fraction (1.2 ml).
Measurement of βgal activity
Cells were grown under the same conditions as those described in "Preparation of bacterial extracts" for the lactose promoter. Five hundred μl of cells were lysed using SDS/chloroform and βgal activity determined and expressed as described in Miller . For Figure 5 and 7, we also prepared soluble extracts using the protocol described in "Preparation of bacterial extracts" and βgal activity was determined as above.
The authors thank Dr S. L. Salhi for help in preparing the manuscript. PM is grateful to Dr. G. Winter for his support and for critical reading of the manuscript. Clones HuLys11 and 225.28S were a kind gift from Dr. Greg Winter and Dr. Dario Neri. This work was supported by funds from the French "Centre National de la Recherche Scientifique".
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