Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli  : A comparison of the synthesis capacities of L-form strains with an E. coli producer strain

Abstract

The paper describes the synthesis of the phosphorylcholine-binding miniantibody McPC603scFvDhl x in cell-wall-less L-form strains of Escherichia coli and Proteus mirabilis. Cells of these strains were transformed with the plasmid pACK02scKan, carrying the miniantibody (miniAb) coding sequence under the control of the lac promoter. L-form transformants of both species were able to synthesize the functional miniAb as an extracellular soluble product. The highest quantities were obtained by P. mirabilis L-form strains after induction with 5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Yields of 45-75 mg/l total antibody protein and of 10-18 mg/l functional miniAb were estimated in the growth medium of shaking cultures 40-80 h after induction with IPTG. About 10% of the active miniAb remained cell-bound. The yields of functional miniAb could be optimized by lowering the growth temperature from 37 degrees C to 26-32 degrees C and by supplementation of the medium with 80 mM sodium fumarate. A comparison of the specific activities revealed that the P. mirabilis L-form strains have a similar synthesis capacity (2-4 mg functional miniAb/g cell dry weight) to that of the producer strain E. coli RV308. The results show that the processes of correct folding and assembling of the miniAb molecules are possible without the periplasmic compartment.

Production of recombinant proteins by high cell density culture of Escherichia coli

“…In another study, fusing the protein (peptide) of interest to the OmpF allowed excretion of the fusion protein into the culture medium when E. coli BL21 host was used (Jeong and Lee, 2002). The use of L-form E. coli strains, which lack in cell walls, is an alternative way of achieving excretory protein production (Kujau et al, 1998). The HCDC of E. coli for the excretory production of recombinant proteins has been reported for limited cases Jeong and Lee, 2002;Kleist et al, 2003).…”

Prokaryotic expression of antibodies

“…These have included bacterial [29,30,[62][63][64][65][66][67], yeast and filamentous fungus [40,68], eukaryotic alga [69], insect cell [70], plant [71], mammalian cell [72,73] and transgenic animal systems [32]. While in many instances rAbs can be expressed in several different expression systems, there is sometimes less flexibility in terms of choice of expression system due to structural requirements on the part of the rAb.…”

Production systems for recombinant antibodies

“…Cell-wall-less L-forms of the Gram-negative bacterium Proteus mirabilis were used for the production of miniAbs and scFvs (35,67), with total and functional scFv yields of 83-127 mg/L and 9 -12 mg/L, respectively (67).…”