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
Jf. Rippmann et al., 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, APPL ENVIR, 64(12), 1998, pp. 4862-4869
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 expre
ssion systems for recombinant antibodies, we compared levels of single-chai
n variable-fragment (scFv) production in Escherichia coli JM109 and P. mira
bilis L VI, which express four distinct scFvs of potential clinical interes
t that show differences in levels of expression and in their tendencies to
form aggregates upon periplasmic expression, production of all analyzed scF
vs in E. call was limited by the severe toxic effect of the heterologous pr
oduct as indicated by inhibition of culture growth and the formation of ins
oluble aggregates in the periplasmic space, limiting the yield of active pr
oduct. In contrast, the L-form cells exhibited nearly unlimited growth unde
r the tested production conditions for all scFvs examined. Moreover, expres
sion experiments with P. mirabilis L VI led to scFv concentrations in the r
ange of 40 to 200 mg per liter of culture medium (corresponding to volume y
ields 33- to 160-fold higher than those with E. coli JM109), depending on t
he expressed antibody, In a translocation inhibition experiment the secreti
on of the scFv constructs was shown to he an active transport coupled to th
e signal cleavage. We suppose that this direct release of the newly synthes
ized product into a Targe volume of the growth medium favors folding into t
he 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 t
he expression process of the host cells, The P, mirabilis L VI supernatant
was found to he advantageous for scFv purification. A two-step chromatograp
hy procedure led to homogeneous scFv with high antigen binding activity as
revealed from binding experiments with eukaryotic cells.