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Biomedical subjects

A Schwienhorst

Publications and source records attributed to A Schwienhorst.

12 recordsLinked to original sources
PubMed2001

A new reporter gene system suited for cell-free protein synthesis and high-throughput screening in small reaction volumes.

The properties of M-hirudin as a new reporter gene system were examined using rabbit reticulocyte lysate for cell-free protein expression. In contrast to the luciferase gene, in vitro translation of M-hirudin is highly robust against changes in concentrations of K+ (and Rb+). In addition, M-hirudin can be detected very sensitively using a reasonably priced fluorimetric thrombin assay. To show that the new reporter gene system is well suited for (u)HTS-applications, cell-free synthesis as well as the fluorimetric assay of M-hirudin were carried out in nanotiter and microtiter plates, respectively.

Animals↗
PubMed2001

Fluorogenic assay for penicillin G acylase activity.

A simple, highly sensitive, and rapid assay for high-throughput screening of penicillin G acylase-producing bacteria is presented. The method is based on the specific release of fluorescent 7-amino-4-methyl-coumarin through cleavage of phenylacetyl-4-methyl-coumaryl-7-amide by penicillin G acylase. The present method is suitable for screening pure enzymes as well as various penicillin G acylases like those from Escherichia coli, Proteus rettgeri, and Kluyvera citrophila in cell extracts. In addition, the new substrate was used for rapid assay of amidase activity in nondenaturing polyacrylamide gels.

Coumarins↗
PubMed2001

Cation radius effects on cell-free translation in rabbit reticulocyte lysate.

The effect of monovalent cation concentrations on the translation was examined in the rabbit reticulocyte cell-free system. The translation of standard reporter gene luciferase was studied using different concentrations of LiCl, NaCl, KCl, RbCl, CsCl, NH(4)Cl, and (CH(3))(4)NCl and the acetates of Na(+), K(+), and NH4(+). Only the salts of K(+), Rb(+), and NH4(+) and to some minor extent of Cs(+) significantly supported translation. Optimum concentrations were dependent on the cation used. Optimum concentrations ranged between 40 mM (NH(4)Ac), 80 mM (KCl, NH(4)Cl), and 100 mM (RbCl, KAc). The maximum efficiency of translation depends on the ionic radius of the cation used. KCl and RbCl were superior to all other salts tested in stimulating in vitro translation. The results were confirmed, using a second reporter system, M-hirudin. Here, however, broad optima were observed with RbCl being slightly superior to KCl in supporting translation.

Animals↗
PubMed2001

High-fidelity in vitro recombination using a proofreading polymerase.

We describe a convenient PCR-based protocol for in vitro recombination of homologous genes, thereby minimizing the rate of associated point mutations. High-fidelity recombination conditions were obtained using Vent DNA polymerase, which, in contrast to Taq DNA polymerase, shows significant proofreading activity and ranges among the slowest thermostable DNA polymerases, allowing tight control of the polymerase-catalyzed DNA extension. To determine the mutagenesis rate and to analyze the efficiency of recombination, 89 clones from a standard experiment were randomly selected for further analysis. Sequence comparison revealed that 21% (19/89) of the clones result from different recombination events in the marker-containing region (260 bp). The overall mutation rate is only 0.02%, which is the lowest rate thus far reported for in vitro recombination experiments.

DNA-Directed DNA Polymerase↗
PubMed2000

Selection of viral RNA-derived tRNA-like structures with improved valylation activities.

The tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) RNA was previously shown to be efficiently charged by yeast valyl-tRNA synthetase (ValRS). This RNA has a noncanonical structure at its 3'-terminus but mimics a tRNA L-shaped fold, including an anticodon loop containing the major identity nucleotides for valylation, and a pseudoknotted amino acid accepting domain. Here we describe an in vitro selection experiment aimed (i) to verify the completeness of the valine identity set, (ii) to elucidate the impact of the pseudoknot on valylation, and (iii) to investigate whether functional communication exists between the two distal anticodon and amino acid accepting domains. Valylatable variants were selected from a pool of 2 x 10(13) RNA molecules derived from the TYMV TLS randomized in the anticodon loop nucleotides and in the length (1-6 nucleotides) and sequence of the pseudoknot loop L1. After nine rounds of selection by aminoacylation, 42 have been isolated. Among them, 17 RNAs could be efficiently charged by yeast ValRS. Their sequence revealed strong conservation of the second and the third anticodon triplet positions (A(56), C(55)) and the very 3'-end loop nucleotide C(53). A large variability of the other nucleotides of the loop was observed and no wild-type sequence was recovered. The selected molecules presented pseudoknot domains with loop L1 varying in size from 3-6 nucleotides and some sequence conservation, but did neither reveal the wild-type combination. All selected variants are 5-50 times more efficiently valylated than the wild-type TLS, suggesting that the natural viral sequence has emerged from a combination of evolutionary pressures among which aminoacylation was not predominant. This is in line with the role of the TLS in viral replication.

3' Untranslated Regions↗
PubMed1998

Novel vector for generating RNAs with defined 3' ends and its use in antiviral strategies.

A novel transcription system was constructed that allows trimming of 3' termini of RNA transcripts in E. coli by endogenous RNase P. Here, the sequence of tRNASer from E. coli fused downstream of the target sequence directs posttranscriptional cleavage 3' of the target sequence. As a first-target MNV11(+), a self-replicating RNA from the QB system was subjected to transcription in vivo. Northern blotting experiments revealed that the primary transcript was indeed successfully processed to an RNA of expected length. The RNA released proved to function as an active template for QB replicase. Moreover, E. coli cells producing these short-chain replicator molecules no longer supported multiplication of QB phages upon infection. Since the novel transcript-trimming system utilizes the endogenous RNase P activity and does not depend on any particular 3'-terminal RNA sequence of target molecules, it may have wide applications for a number of different targets in prokaryotes. Further applications, including those in eukaryotes, are discussed.

Antiviral Agents↗
PubMed1997

Display of functional thrombin inhibitor hirudin on the surface of phage M13.

A synthetic gene for hirudin was ligated into phagemid pCANTAB5E. This construct allows production of either soluble hirudin or phage having hirudin displayed on the surface. Similarly, hirudin variants with extensions either at their N- or C-terminus were generated. The genes were expressed in their soluble form in a non-suppressor strain of E. coli. Periplasmatic fractions were evaluated in standard thrombin inhibition assays. Extending hirudin by a single Gln residue at the N-terminus reduces the activity by two orders of magnitude. This suggests that either the terminal amine group makes an important interaction or that steric constraints do not allow additional amino acids here. Only C-terminal extensions maintain most of the thrombin inhibitor activity of r-hirudin. The r-hirudin gene was also expressed on the tips of filamentous phage as a fusion protein with protein III (pIII). The hirudin-pIII fusion protein was detected with anti-hirudin antibody and with anti-E-tag antibody by Western blot analysis. Recombinant phages were shown to bind to immobilized thrombin in a dose-dependent manner. Upon addition of soluble thrombin, recombinant hirudin phages could be eluted specifically. Finally, purified phages carrying displayed r-hirudin were shown to inhibit thrombin in a standard amidolytic assay for thrombin inhibitor activity. These results demonstrate that hirudin can be C-terminally extended without diminishing the antithrombic activity. Beyond that, active hirudin can be displayed on the surface of M13 phage. As a conclusion, applied molecular evolution, i.e. the selection of hirudin-based thrombin inhibitor variants with tailored properties from (partially) randomized peptide pools should now be possible.

Bacteriophage M13↗
PubMed1997

Rapid selection of aminoacyl-tRNAs based on biotinylation of alpha-NH2 group of charged amino acids.

A rapid selection procedure to separate low amounts of aminoacylated tRNAs from large pools of inactive variants is described. The procedure involves a three-step protocol. After initial aminoacylation of a tRNA pool, N-hydroxysuccinimide ester chemistry is applied to biotinylate the alpha-NH2 group of the amino acid bound to the 3'-end of a tRNA. The biotin tag is used to capture the derivatized tRNAs on streptavidin-conjugated magnetic beads. Variants bound to the solid phase can be amplified by RT-PCR and transcription, providing tRNAs for subsequent selection rounds.

Amino Acids↗
PubMed1997

Optimizing doped libraries by using genetic algorithms.

The insertion of random sequences into protein-encoding genes in combination with biological selection techniques has become a valuable tool in the design of molecules that have useful and possibly novel properties. By employing highly effective screening protocols, a functional and unique structure that had not been anticipated can be distinguished among a huge collection of inactive molecules that together represent all possible amino acid combinations. This technique is severely limited by its restriction to a library of manageable size. One approach for limiting the size of a mutant library relies on 'doping schemes', where subsets of amino acids are generated that reveal only certain combinations of amino acids in a protein sequence. Three mononucleotide mixtures for each codon concerned must be designed, such that the resulting codons that are assembled during chemical gene synthesis represent the desired amino acid mixture on the level of the translated protein. In this paper we present a doping algorithm that "reverse translates' a desired mixture of certain amino acids into three mixtures of mononucleotides. The algorithm is designed to optimally bias these mixtures towards the codons of choice. This approach combines a genetic algorithm with local optimization strategies based on the downhill simplex method. Disparate relative representations of all amino acids (and stop codons) within a target set can be generated. Optional weighing factors are employed to emphasize the frequencies of certain amino acids and their codon usage, and to compensate for reaction rates of different mononucleotide building blocks (synthons) during chemical DNA synthesis. The effect of statistical errors that accompany an experimental realization of calculated nucleotide mixtures on the generated mixtures of amino acids is simulated. These simulations show that the robustness of different optima with respect to small deviations from calculated values depends on their concomitant fitness. Furthermore, the calculations probe the fitness landscape locally and allow a preliminary assessment of its structure.

Algorithms↗
PubMed1996

Hamming chromatography.

Selection of molecules with desired properties from random pools of biopolymers has become a powerful tool in biotechnology. On designing an evolution experiment, a certain knowledge of the concomitant fitness landscape is clearly helpful to set up the optimal experimental conditions. The correlation function is a useful means of characterizing a given landscape, since it can be efficiently measured if one has a method of separating a pool of random sequences according to their Hamming distance from a moderately small number of test sequences. In this paper we describe a special type of hybridization chromatography, where a mixture of oligomers (partially) complementary to a given test sequence is hybridized to the test sequence, covalently bound to a matrix. DNA oligomers are eluted in an 'effective temperature gradient' using conditions that minimize the differences of effects of GC versus AT pairs on the melting temperatures. This method should be a means to quickly separate error classes and thus be the crucial step in characterizing fitness landscapes of biopolymers through an experimental approach. It would also be a useful tool to design sequence pools with a bias towards desired mutant spectra.

Bacteriophage T7↗
PubMed1993

Accurate high-speed liquid handling of very small biological samples.

Molecular biology techniques require the accurate pipetting of buffers and solutions with volumes in the microliter range. Traditionally, hand-held pipetting devices are used to fulfill these requirements, but many laboratories have also introduced robotic workstations for the handling of liquids. Piston-operated pumps are commonly used in manually as well as automatically operated pipettors. These devices cannot meet the demands for extremely accurate pipetting of very small volumes at the high speed that would be necessary for certain applications (e.g., in sequencing projects with high throughput). In this paper we describe a technique for the accurate microdispensation of biochemically relevant solutions and suspensions with the aid of a piezoelectric transducer. It is suitable for liquids of a viscosity between 0.5 and 500 milliPascals. The obtainable drop sizes range from 5 picoliters to a few nanoliters with up to 10,000 drops per second. Liquids can be dispensed in single or accumulated drops to handle a wide volume range. The system proved to be excellently suitable for the handling of biological samples. It did not show any detectable negative impact on the biological function of dissolved or suspended molecules or particles.

Base Sequence↗