Directed evolution of single-chain Fv for cytoplasmic expression using the beta-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation

Abstract

BACKGROUND: 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. RESULTS: We used the beta-galactosidase alpha-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 beta-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 beta-galactosidase activity present in the cells. CONCLUSION: 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 alpha-peptide released from the protein fusion by the host proteases. This means that the alpha-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released alpha-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.

Figure 1

Schematic view of plasmid pPM170 Plasmid pPM170 allows the cloning of scFv genes between the NcoI and NotI sites under the control of the lactose promoter. The translated protein is fused at its C-terminal extremity to the α-fragment of βgal. A) Plasmid map with the main restriction sites; B) DNA and aminoacid sequences fused to the 3' end and the C-terminus of the cloned scFv. The scFv sequence is followed by two tags (in blue), the α-fragment of βgal (in green, aminoacids 6 to 59 of βgal) and a 30 aminoacid long peptide (in orange) originating from pBR322 plasmid and M13 gene IV sequence (see text). The sequences of the two sequenced spontaneous lac+ mutants, 173S1 and 173S2 (see text), are also shown.

Figure 2

Lactose phenotype correlates with the soluble expression levels Phenotype of strain TG1 transformed with the pPM170 plasmid containing a series of scFv with different soluble expression levels in E. coli cytoplasm. The scFv13, 13R1, 13R2, 13R3 and 13R4 are described in [28]. T- and T+ are TG1 transformed with the pTrc99A and pUC119 plasmids, respectively. 173S1 is a spontaneous lac+ mutant isolated from TG1(pPM173) (see text and Fig. 1). A) Lactose phenotype on MacConkey lactose plates. The ability of the cell to use lactose is proportional to the depth of the red coloration. B) βgal activity present in the cytoplasm of the cells measured using Miller's whole cell assay [30].

Figure 3

Effect of the 30 aminoacid long C-terminal peptide on scFv expression level scFv13 and scFv13R4 were cloned either in pPM170 (173 and 173R4) or in pPM173S1 (173S1 and 173S1-R4), and expressed in strain TG1. A) Phenotype of the strains on MacConkey lactose plates after 24 h at 37°C. B) Soluble and insoluble extracts of the four strains were analyzed by Coomassie blue staining (top) or by western blot (bottom) using the 9E10 anti-c-myc monoclonal antibody followed by an anti-mouse HRP-conjugated antibody and detected with a commercial kit (Pierce Supersignal West Pico kit #14079). The arrows show the position of the fusions (38 kDa for 173 and 173R4; 34 kDa for 173S1 and 173S1-R4) and the unfused scFv (28 kDa).

Figure 4

Outline of the selection procedure A) Outline of the steps followed during the selection procedure; B) Size of the libraries generated and number of clones isolated after each round of selection and used as a pool for the next round.

Figure 5

Phenotype of the isolated mutants After four rounds of mutagenesis and selection, the five best clones of scFv 225.28S were analyzed in strain TG1. pPM175 contains the wild type scFv and pPM175R4.1 to pPM175R4.5 the five isolated mutants. T+ and T- are the same as in Fig. 2. A) Phenotype of the strains on MacConkey lactose plates; B) βgal activity measured as in Figure 1. In addition to the Miller's whole cell assay (blue), we analyzed in B the βgal activity present in soluble extracts (red).

Figure 6

Location of isolated mutations in HuLys11 scFv structure Schematic representation of the HuLys11 scFv (pdb 1BVK). The CDR loops are represented in blue. The side chain of three of the four isolated mutations (see additional file HuLys11aa.txt) are represented in red. The fourth mutation, located in the linker sequence, is not present in the structure.

Figure 7

Expression of scFv-α fusions in TG1 and TG1lon strains SDS-PAGE of soluble (5 μl : A, C & F) and insoluble extracts (5 μl ; B, D & G) of the five best clones of scFv 225.28S (A, B, C & D) and scFv HuLys11 (F & G) expressed in TG1 and TG1lon. Proteins were revealed either by Coomassie blue staining (A & B) or by western blot (C, D, F & G) using an HRP-coupled anti-polyHistidine monoclonal antibody (Sigma A7058) and detected with a commercial detection kit (Pierce Supersignal West Pico kit #14079). In panel E, the same soluble extracts than in C were tested for βgal activity.

Figure 8

Cytoplasmic expression of unfused scFv After cloning in plasmid pPM210, scFv225.28S and mutants were expressed in strain TG1lon. 5 μl of soluble and insoluble extracts were analyzed by Coomassie blue staining (A) and western blot (B) using the 9E10 anti-c-myc monoclonal antibody followed by an anti-mouse alkaline phosphatase-conjugated antibody and detected with the chromogenic substrate BCIP/NBT.

Figure 9

Comparison of cytoplasmic and periplasmic soluble expression Genes of scFv225.28S and its mutant R4.1 were cloned in plasmid pAB1 [28]. In this plasmid, scFv genes are expressed under the control of the lac promoter with the pelB signal sequence fused at their N-terminal extremity in order to target protein to the periplasm. Soluble and insoluble extracts were prepared from strain TG1 and were analyzed by Coomassie blue staining (A) and western blot (B) as in Figure 8. The two last extracts (Cyto) are those analyzed in lanes 1 and 2 of Figure 8 (soluble cytoplasmic extracts of scFv225.28S and R4.1, cloned in pPM210 and expressed in TG1lon).

Figure 10

Cytoplasmic expression under the T7 promoter SDS-PAGE of soluble and insoluble extracts (5 μl) of the best clones isolated after four rounds of mutagenesis, cloned in the pET23NN plasmid. Proteins were revealed either by Coomassie staining (top) or by western blot using the 9E10 anti-c-myc monoclonal antibody followed by an anti-mouse alkaline phosphatase-conjugated antibody and detected with the chromogenic substrate BCIP/NBT. A) HuLys11 scFv and mutants; B) 225.28S scFv and mutants.

Figure 11

Schematic representation of the possible in vivo fates of scFv-α protein fusion Schematic representation of folding, aggregation and degradation processes. In the scheme presented, the newly translated protein (left) is assumed to proceed through two alternative pathways: either it folds to give the folded protein (soluble native) or it evolves via a side reaction leading to a misfolded protein (misfolding). A second kinetic competition is thought to occur between degradation and aggregation of the misfolded protein. Green arrows represent the kinetic competition between folding and aggregation [44] on which the soluble reporter assays are based [18]. Red arrows represent an additional pathway leading to degradation [43] and release of free α-peptide in the cytoplasm.