Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli

Abstract

Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

FIG. 1

(A) Growth kinetics of E. coli JM109(pEA11) scFv H398 antigen binding activity. (B) Western blot analysis of the soluble and unsoluble proteins of E. coli JM109(pEA11) lysates. Cells were cultivated in 20 ml of medium (BHI-yeast extract-kanamycin) at 30°C, and scFv H398 synthesis was induced at an OD₅₅₀ of 0.8 with 0.5 mM IPTG. At different times after induction of scFv synthesis, cells were harvested, sonicated, and fractionated by centrifugation at 10,000 × g for 10 min. The amount of active scFv H398 in the soluble protein fraction was determined by ELISA. Western blot analysis was performed as described in Materials and Methods with 12 μg of soluble cell protein or the corresponding volume of the insoluble protein suspension per lane of an SDS–15% polyacrylamide gel. The primary detection antibody was anti-c-myc MAb. ▵, uninduced culture of JM109(pEA11); ▿, induced culture of JM109(pEA11); •, active scFv H398 determined by ELISA. Molecular masses (in kilodaltons) of marker proteins are indicated at the right of the gel.

FIG. 2

(A) Growth kinetics of P. mirabilis L VI(pEA11) and scFv H398 antigen binding activity. (B) Western blot analysis of the cell pellet and the culture supernatant. Cells were cultivated, harvested and fractionated as described in the legend to Fig. 1. The amount of active scFv H398 in the supernatant was determined by ELISA. Western blot analysis was performed as described in Materials and Methods with the cell suspension at an OD₅₅₀ of 0.03 (8 μg of soluble cell protein) and with a corresponding volume of the supernatant in the lanes of an SDS–15% polyacrylamide gel. The primary detection antibody was anti-c-myc MAb. ▵, uninduced culture of L VI(pEA11); ▿, induced culture of L VI(pEA11); •, active scFv H398 determined by ELISA. Molecular masses (in kilodaltons) of marker proteins are indicated at the right of the gel.

FIG. 3

Inhibition of the scFv H398 secretion by sodium azide. scFv H398 synthesis was induced in a 20-ml shaker-flask culture of P. mirabilis LVI(pEA11) by the addition of 0.5 mM IPTG. After two generations (2.5 h), the translocation of the preprotein was inhibited by the addition of sodium azide at a final concentration of 0.02% (wt/vol). Culture aliquots (0.5 ml) were harvested at the indicated times after addition of sodium azide, and the cells were separated from the supernatant by centrifugation (10,000 × g, 10 min). Western blot analysis of the cell fraction and the supernatant was performed as described in Materials and Methods. The primary detection antibody was either anti-c-myc 9E10 or serum B, as indicated above the lanes. Molecular masses (in kilodaltons) of marker proteins are indicated at the right. c., cell fraction; s., culture supernatant; p, premature scFv H398; m, mature scFv H398.

FIG. 4

Aggregation of scFv H398 in culture supernatant. (A) Antigen binding activity. (B) Western blot analysis of aggregated scFv H398. The supernatant of an induced overnight culture of L VI(pEA11) was further incubated at 30°C under agitation with or without nonproducing P. mirabilis L VI(pACK02scKan-Δab) cells. Samples of the cell suspension or the supernatant were taken at indicated times and fractionated by centrifugation (10,000 × g, 10 min). The activity of scFv H398 in the soluble fraction was determined by ELISA. For the Western blot analysis, 5 μl of the soluble fraction and a corresponding amount of the pellet fraction from the incubation with cells were separated on an SDS–15% polyacrylamide gel. ▵, supernatant incubated without cells; ○, supernatant incubated with P. mirabilis LVI(pACK02scKan-Δab). Molecular masses (in kilodaltons) of marker proteins are indicated at the right of the gel.

FIG. 5

Purification of scFv H398 protein by SDS–12% PAGE in combination with Coomassie blue staining was used for analysis of scFv H398-containing samples. Lane 1 shows low-molecular-mass markers. Lanes 2 to 4 were loaded with 20-μl samples of the cell fraction, the culture supernatant with 100 mg of scFv H398 per liter and the flowthrough of the IMAC, respectively. Lane 5 was loaded with 4 μl of the pooled IMAC eluate fractions. Scanning of lane 5 revealed a purity of more than 90% for scFv H398, which is visible as a 31-kDa protein. Molecular masses (in kilodaltons) of marker proteins are indicated at the left.

FIG. 6

Plot of the results of analytical size exclusion chromatography of scFv H398. A 50-μl aliquot of the pooled IMAC fraction was loaded on a Superdex 75 column and analyzed by the SMART Manager version 1.31 standard protocol. Fractions of 80 μl were collected, and binding activity was analyzed by ELISA with TNFR60 as the antigen. Total scFv H398 content was determined by direct coating of the ELISA plates with the fractions. Standard proteins were BSA (67 kDa) and chymotrypsinogen (24 kDa), which eluted with fractions 9 and 13, respectively. ○, scFv H398 amount; hatched bars, antigen binding activity of scFv H398.

FIG. 7

Binding activity of scFv OS4 to FAP⁺ and FAP⁻ HT1080 cells. Aliquots of purified scFv OS4 after IMAC or after consecutive size exclusion chromatography were diluted to give 10- or 4-ng/μl concentrations of scFv OS4, respectively, in PFA or PBS plus milk (2%). Dilutions were chosen to give comparable signals of milk-treated IMAC and size exclusion chromatography purified samples. The scFv OS4-containing samples (75 μl) were incubated with 10⁴ HT1080 cells with or without FAP on their cell surfaces. Specific and unspecific binding of scFv OS4 was monitored with anti-c-myc antibody as the primary detection antibody. Error bars indicate the standard errors of the means (n = 3).