Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates.

Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.

An optical method for the detection of oxidative stress using protein-RNA interaction.

The cytosolic 4Fe-4S protein aconitase can be converted under the influence of reactive oxygen species into an iron-regulatory protein (IRP1). Therefore, the IRP1 level is considered as an indirect marker of oxidative stress. An experimental approach is presented here to detect the concentration of this marker protein by surface plasmon resonance. The optical method exploits the natural binding affinity of IRP1 to an iron-responsive element (IRE) which was in vitro transcribed with a linker sequence and subsequently immobilized on a BIACORE sensor chip. The detection was found to be reproducible and sensitive in the range 20-200 nM IRP. Conditions of the binding process, such as pH and thiol concentration, were characterized. Feasibility of the method to detect and quantify IRP1 in physiological media was demonstrated.

Development of a creatinine ELISA and an amperometric antibody-based creatine sensor with a detection limit in the nanomolar range.

Creatinine-specific antibodies have been generated and used for highly sensitive and specific immunochemical creatinine determinations. Creatinine was derivatized at N3 and coupled to KLH carrier protein. On the basis of this immunogen, monoclonal antibodies were developed by hybridoma technology. Antibodies from various clones have been characterized with BIAcore 2000 with respect to the dissociation constant and specificity. Antibodies of clone B90-AH5 exhibited the lowest dissociation constant (0.74 microM) and the highest specificity for creatinine and were chosen for the development of a competitive ELISA and an amperometric creatinine sensor. The creatinine sensor was constructed by fixing a creatinine-modified membrane on the top of a platinum working electrode which was then incorporated into a stirred electrochemical measuring cell. For creatinine determination the creatinine-containing sample was incubated with B90-AH5 and anti-IgG(mouse)-glucose oxidase conjugate and applied to the measuring cell. After a washing step glucose was added and the produced hydrogen peroxide was registered at Eappl = +600 mV vs Ag/AgCl. The measuring range was 0.01-10 microg/mL. The highest sensitivity for creatinine was achieved at 330 ng/mL (3 microM) and the lower detection limit at 4.5 ng/mL (40 nM). This is far below the relevant clinical range, which is 5-17 microg/mL (44-150 microM) and allows a reliable determination of very low creatinine concentrations in serum, where standard methods cannot be applied. After each measurement the sensor was regenerated with 10 mM HCl without any loss in binding activity.

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody.

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.

Molecular basis for the binding promiscuity of an anti-p24 (HIV-1) monoclonal antibody.

Multiple binding capabilities utilized by specific protein-to-protein interactions in molecular recognition events are being documented increasingly but remain poorly understood at the molecular level. We identified five unrelated peptides that compete with each other for binding to the paratope region of the monoclonal anti-p24 (HIV-1) antibody CB4-1 by using a synthetic positional scanning combinatorial library XXXX[B1,B2,B3,X1,X2,X3]XXXX (14 mers; 68,590 peptide mixtures in total) prepared by spot synthesis. Complete sets of substitution analogs of the five peptides revealed key interacting residues, information that led to the construction of binding supertopes derived from each peptide. These supertope sequences were identified in hundreds of heterologous proteins, and those proteins that could be obtained were shown to bind CB4-1. Implications of these findings for immune escape mechanisms and autoimmunity are discussed.

Enzymes and antibodies in organic media: analytical applications.

This chapter outlines the influence of organic solvents on antibodies, enzymes, and their reactions, the different kinds of enzyme-solvent systems and the various advantages of organic solvents compared with water, with respect to analytical purposes. Examples for electrochemical, optical and thermometric assays in organic solvents are given. The potential of organic solvents for the modification of immunoassays is exemplified, opening up new applications.

Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus.

By screening of 15 thermophilic Bacillus strains, five strains exhibiting lipase activity were found. Among these the strain Bacillus thermocatenulatus (DSM 730) produced the highest lipase activity. The lipase proved to be inducible and extracellular and was purified 67-fold to homogenous state by hexane extraction, methanol precipitation and ion-exchange chromatography on Q-Sepharose. The molecular weight of the lipase determined by SDS-PAGE is 16 kDa. However, the lipase forms very large aggregates (> 750 kDa) as observed after native PAGE, which makes handling of the lipase very difficult. The lipase binds almost irreversibly on different chromatography matrices, e.g., Amberlite and Serolite, and is very stable in the immobilised form. The N-terminal sequence consists of 53% apolar amino acids and shows no significant homology towards other known lipase sequences. Maximum activity was found at pH 7.5-8.0 and 60-70 degrees C with pNPP and olive oil as substrates.

Purification and properties of a lipase from Penicillium expansum.

Penicillum expansum DSM 1994 produces a new, inducible extracellular lipase when grown in medium containing 0.1% olive oil. Maximum activity was obtained after 4 days of incubation at 20 degrees C. The enzyme was purified 219-fold by cross-flow filtration, ammonium sulfate precipitation and hydrophobic interaction chromatography to a final specific activity of 558 U/mg. The molecular weight of the homogeneous lipase was (25 kDa) determined by gel filtration and SDS-PAGE, however, it forms active dimers and higher aggregates as observed after native PAGE. The enzyme was identified as a glycoprotein with a pI of 5.5. The N-terminal sequence shows a homology to sequences of other lipase just behind their consensus sequence. Enzyme stability was enhanced by the addition of Tween 20 and Lubrol PX. The enzyme showed a maximum activity at pH 9 at 45 degrees C and was stable at a broad pH range of 6-10. Lipase of P. expansum showed a preference for triacylglycerols, but no positional specificity.

Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis.

An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography procedure. The enzyme is metal-free and has a molecular mass of about 51 000 Da and probably consists of a single polypeptide chain. As shown by fluorimetric titration, the prosthetic group is 1 mol FAD/mol protein. The affinity behaviour of the enzyme gives evidence for the existence of a dinucleotide-binding domain capable of binding NADH or FAD. The enzyme is specific for NADH (Km = 4.1 X 10(-5) M), NADPH is not oxidized. O2 is the preferred electron acceptor, in addition FAD and, very slowly, one-electron acceptors are reduced. It is not clear whether the reduction of FAD proceeds through the dinucleotide-binding site or by exchange of the prosthetic group. The stoichiometry of the reaction with O2 corresponds to the consumption of 2 mol NADH/mol O2, and only H2O is formed (2 NADH + 2 H+ + O2----2 NAD+ + 2 H2O). Neither H2O2 nor O2.- is detected as intermediate and H2O2 cannot replace O2 as an oxidant. The enzyme can, mainly in its reduced state, be inhibited by -SH reagents. Spectral data give no evidence for the existence of radical intermediates during reduction. The enzyme can obviously accept more than two electrons/mol. On the basis of these data two possible reaction mechanisms are discussed. A proposal for the biological purpose of the reaction is made.

Multiple allelic states of the cyh2 gene cause low- and high-level cycloheximide resistance in Saccharomyces cerevisiae.

At least four different mutations at the cyh2 locus (rp1X; gene product: YL24) of Saccharomyces cerevisiae confer cycloheximide resistance. The mutant YL24 proteins are either more basic (high-level resistant phenotype), more acidic (low-level resistant phenotype), or unchanged in their electrophoretic mobility (both low-and high-level resistant phenotypes). None of the mutations at other loci seem to induce high-level resistance to cycloheximide.

Binding of cycloheximide to ribosomes from wild-type and mutant strains of Saccharomyces cerevisiae.

Cycloheximide bound to cytoplasmic (80S) ribosomes of the yeast Saccharomyces cerevisiae with an association constant (Ka) of 2.0 (+/- 0.5) x 10(7) M-1. The number of binding sites found per ribosome was between 0.4 and 0.6; it was reduced by high-salt treatment of ribosomes 60S particles prepared in the presence of high salt had a lower affinity (Ka: 5.5 [+/- 0.5] x 10(6) M-1) than did 80S ribosomes, but a greater proportion of particles (0.8) were able to bind. No specific binding to 40S subunits was observed. The addition of supernatant fractions (S100, high-salt wash fraction) increased the number of binding sites found per 80S ribosome up to 0.8, leaving the association constant unchanged. In contrast, the affinity of 60S subunits was enhanced to a Ka value of 3.5 x 10(-7) M-1 by the addition of supernatant fractions, whereas the number of binding sites stayed constant. A model to explain these facts is proposed. 80S ribosomes, as well as 60S subunits of strain cy32, which is highly resistant to cycloheximide and altered in ribosomal protein L29 (18), showed a drastically reduced affinity for the drug (Ka values of 2.0 x 10(6) M-1).

Altered ribosomal protein L29 in a cycloheximide-resistant strain of Saccharomyces cerevisiae.

A spontaneous high-level cycloheximide-resistant mutant of the yeast Saccharomyces cerevisiae (strain cy32) is found to have an altered protein of the large subunit (60S) of cytoplasmic ribosomes, namely protein L29. The resistance character segregates together with this biochemical defect and is semidominant in heterozygous diploids. Judged from in vitro susceptibility to inhibition by cycloheximide there are at least 50% resistant ribosomes present in such diploid strains. From these results it is concluded that cycloheximide resistance of mutant cy32 is caused by mutation of a single gene and that it is the structural gene for L29 which is affected.Preliminary genetic mapping data are also reported. They indicate a location of cyhx-32 marker on chromosome 7 near met13.