[Isolation of glutamylendopeptidase precursor from B. licheniformis and its processing in vitro]. / Poluchenie predshestvennika glutamiléndopeptidazy B. licheniformis i izuchenie ego protsessinga in vitro.

A secretory system based on L-form cells of Proteus mirabilis was developed for production of native Bacillus licheniformis glutamylendopeptidase precursor never formerly available. The produced precursor was stable per se under physiological conditions and in presence of trypsin and glutamylendopeptidase from B. intermedius. Complete conversion of the precursor to the mature glutamylendopeptidase was performed by bacillar metalloproteases and subtilisin. The artificially processed glutamylendopeptidase was purified by affinity chromatography on bacitracin-sepharose. Native tertiary structure in the purified glutamylendopeptidase was confirmed by demonstrating its activity towards a specific chromogenous peptide substrate.

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.

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.

High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions.

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl-1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hoklsok suicide system, improving plasmid maintenance, and the inducible lac pl o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l-1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fedbatch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 gl-1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

Formation of recombinant proteins in Escherichia coli under control of a nitrogen regulated promoter at low and high cell densities.

The use of a modified Escherichia coli glnAP2 promoter results in the formation of both homologous and heterologous, cytoplasmic and periplasmic recombinant proteins in a nitrogen concentration dependent manner. As in the E. coli nitrogen regulatory system, glnAP2 controlled gene expression is induced when ammonium concentration in the growth medium is below 1 mM (nitrogen limitation), otherwise only extremely low expression of recombinant genes is observed. Both high cell density cultivations (HCDC) and low cell density cultivations (LCDC) gave similar results for inducibility and formation of the following recombinant proteins: chloramphenicol-acetyltransferase, phosphorylcholine binding mini-antibodies (scFv-dhlx of McPC603) and K1-streptokinase. Recombinant proteins were formed in quantities of about 2-3% of total cellular protein. At low cell densities, slightly higher quantities resulted under partial nitrogen limitations than under total nitrogen limitation. In contrast, high cell density cultivations resulted in lower product concentrations at partial nitrogen limitation compared with total nitrogen limitation. These lowered product concentrations were probably due to the very high amounts of K+ or Na+ ions which accumulated during pH-regulation, thereby disturbing growth. HCDC under partial nitrogen limitation decreased proteolysis of recombinant proteins, therefore reduced amounts of proteases are considered to be responsible.

Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli.

The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli. For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form "miniantibodies" with significantly higher avidity than in the case of leucine zipper containing constructs. The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken. The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment. We report here a high-cell density fermentation of E. coli producing these miniantibodies and a work-up procedure suitable for large scale production. Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E. coli culture.

Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase.

The expression of the glyoxylate cycle enzymes is required for growth of the yeast Yarrowia lipolytica on acetate or fatty acids as sole carbon source. Acetyl-coenzyme A, which is produced by acetyl-coenzyme A synthetase (ACS) from acetate, is needed for induction of this expression. Acetate-non-utilizing mutants of this yeast were investigated in order to identify mutants which express no or strongly reduced activity of this enzyme. Mutations in gene ICL2 exhibited the strongest effects on the activity. In icl2 mutants, lack of ACS activity resulted in a non-induced glyoxylate cycle on acetate; however, induction on fatty acids was not affected. Gene ICL2 was identified as the structural gene encoding the monomer of ACS. It is shown that a high level of ACS activity is necessary for full expression of the glyoxylate cycle enzymes. Mutations in gene ICL1, which encodes isocitrate lyase, resulted in overproduction of ACS without any growth on acetate. A new gene (GPR1 = glyoxylate pathway regulation) was detected in which trans-dominant mutations inhibit expression of ACS and the glyoxylate cycle on acetate as carbon source.