Co-operative effects of protein engineering and vector optimization on high yield expression of functional bivalent miniantibodies in Escherichia coli.

The volumetric yield of functional phosphocholine-binding miniantibodies could be increased in E. coli fermentations by the combination of the following approaches: Firstly, miniantibody mutants with amino acid exchanges in the VH chain leading to improved folding were expressed. Secondly, the expression vector was stabilized by an efficient suicide system to prevent plasmid loss. Thirdly, the cells were grown to high cell densities in a stirred tank reactor.

Intramolecular derivatization of 2'-amino-pyrimidine modified RNA with functional groups that is compatible with re-amplification.

To expand the scope of nucleic acid aptamers as a tool for precise molecular recognition, functional groups that are not naturally present in nucleic acid molecules are desired. For in vitro selection these new functional groups must be compatible with the selection process. The present method allows the introduction of succinimide activated side chains at internal amino groups of 2'-amino-pyrimidine-RNA in a combinatorial fashion that is compatible with enzymatic re-amplification.

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.

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.

Efficient preparation of single-stranded DNA for in vitro selection.

In vitro selection of aptamers requires the reliable enzymatic preparation of large amounts of (+) single-stranded DNA molecules. This can be achieved by selective enzymatic digest of 5'-phosphorylated (-) strands from PCR products, a method already widely used in sequencing of PCR products. Here we present an adaptation of this method to prepare large pools of single-stranded DNA molecules for in vitro selection.

Design of leader sequences that improve the efficiency of the enzymatic synthesis of 2'-amino-pyrimidine RNA for in vitro selection.

The application of nucleic acids obtained by in vitro selection from a large pool of molecules with random sequences in medical diagnosis or therapy requires nucleic acids with enhanced stability in biological fluids. Chemical modifications introduced after selection are likely to alter the structure and the properties of the selected molecules. Therefore, the chemical modifications used must be present throughout the selection. This can be achieved for example by the incorporation of 2'-amino-pyrimidine nucleotides into RNA in the transcription step. Though modified molecules could be transcribed from some generally designed dsDNA templates, the efficiency of transcription and reverse transcription and reverse transcription was very low making this strategy too inefficient. Templates and primers with varying amounts of pyrimidines in the constant flanking region of the RNA molecule were designed and their efficiency in transcription and reverse transcription tested. The obtained 2'-amino-pyrimidine RNA molecules showed enhanced stability in serum and RNAse cocktails. Here we present optimized leader sequences flanking the random core-sequence and reaction conditions that allow the reliable utilization of this modification in in vitro selection.