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M Ostermeier

Publications and source records attributed to M Ostermeier.

14 recordsLinked to original sources
PubMed2001

Creating multiple-crossover DNA libraries independent of sequence identity.

We have developed, experimentally implemented, and modeled in silico a methodology named SCRATCHY that enables the combinatorial engineering of target proteins, independent of sequence identity. The approach combines two methods for recombining genes: incremental truncation for the creation of hybrid enzymes and DNA shuffling. First, incremental truncation for the creation of hybrid enzymes is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers. SCRATCHY libraries were created from the glycinamide-ribonucleotide formyltransferase (GART) genes from Escherichia coli (purN) and human (hGART). The developed modeling framework eSCRATCHY provides insight into the effect of sequence identity and fragmentation length on crossover statistics and draws contrast with DNA shuffling. Sequence analysis of the naive shuffled library identified members with up to three crossovers, and modeling predictions are in good agreement with the experimental findings. Subsequent in vivo selection in an auxotrophic E. coli host yielded functional hybrid enzymes containing multiple crossovers.

Algorithms↗
PubMed2001

Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides.

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

Amino Acid Sequence↗
PubMed2000

A two-phagemid system for the creation of non-phage displayed antibody libraries approaching one trillion members.

We have designed a two-phagemid system for the construction of very large non-phage displayed Fab antibody libraries in E. coli approaching 10(12) members. The system can accommodate both periplasmic and cytoplasmic Fab expression and should prove useful for the direct selection of functional antibodies by genetic techniques. We successfully alleviate problems of Fab vector instability and report a set of improved 5' primers for the amplification of mouse Ig V(H)95% of mouse Ig V(H) genes and minimize the amount of N-terminal amino acid changes while maintaining the flexibility of periplasmic or cytoplasmic antibody expression in E. coli.

Amino Acid Sequence↗
PubMed2000

Autonomic nervous system-controlled cardiac pacing: a comparison between intracardiac impedance signal and muscle sympathetic nerve activity.

A recently introduced rate responsive cardiac pacing system is based on information derived from the intracardiac impedance signal containing information on the inotropic state of the ventricle. This study compared the inotropic state index (ISI) with muscle sympathetic activity (MSA), both being modulated by the autonomic nervous system. Nine patients (66 +/- 3 years, mean +/- SEM) with Inos2DR pacemakers were included. Each patient was studied at rest and during cold pressor test (CPT). Microneurography of the peroneal nerve was performed to measure MSA continuously, which was digitally stored along with continuous surface ECG and blood pressure. The intracardiac impedance signal was transmitted by the pacemaker and stored simultaneously. Linear correlation between ISI and MSA was calculated for the period of the CPT. During CPT, mean systolic blood pressure increased from 122 +/- 4 to 149 +/- 6 mmHg (P < 0.0001), diastolic blood pressure increased from 74 +/- 8 to 86 +/- 4 mmHg (P = 0.02), and intrinsic heart rate increased from 69 +/- 7 to 75 +/- 7 beats/mill (P = 0.019). ISI increased by 21 +/- 7% (P = 0.018), MSA by 26 +/- 6% (P = 0.004). ISI and MSA were positively correlated during the CPT in eight of nine patients (R2 = 0.86-0.99, P < 0.0001). Negative correlation was found in one patient (R2 = 0.94). This study demonstrates parallel increases of the ISI and MSA during CPT. ISI and MSA showed a close linear relationship during provoked changes of sympathetic activity. These results provide further evidence that the sympathetic nervous system is responsible for the observed ISI changes.

Aged↗
PubMed1999

Combinatorial protein engineering by incremental truncation.

We have developed a combinatorial approach, using incremental truncation libraries of overlapping N- and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer. This general method for enzyme bisection will have broad applications in the engineering of new catalytic functions through domain swapping and chemical synthesis of modified peptide fragments and in the study of enzyme evolution and protein folding. We have tested this methodology on Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and, by genetic selection, identified PurN heterodimers capable of glycinamide ribonucleotide transformylation. Two were chosen for physical characterization and were found to be comparable to the wild-type PurN monomer in terms of stability to denaturation, activity, and binding of substrate and cofactor. Sequence analysis of 18 randomly chosen, active PurN heterodimers revealed that the breakpoints primarily clustered in loops near the surface of the enzyme, that the breaks could result in the deletion of highly conserved residues and, most surprisingly, that the active site could be bisected.

Amino Acid Sequence↗
PubMed1999

Incremental truncation as a strategy in the engineering of novel biocatalysts.

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.

Amino Acid Motifs↗
PubMed1999

A combinatorial approach to hybrid enzymes independent of DNA homology.

We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries between genes in a manner that is independent of DNA homology. We compared the ability of ITCHY and DNA shuffling to create interspecies fusion libraries between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, which have only 50% identity on the DNA level. Sequencing of several randomly selected positives from each library illustrated that ITCHY identified a more diverse set of active fusion points including those in regions of nonhomology and those with crossover points that diverged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were greater than or equal to the activity of the hybrids found by DNA shuffling.

Amino Acid Sequence↗
PubMed1998

Hybrid enzymes: manipulating enzyme design.

Hybrid enzymes are engineered to contain elements of two or more enzymes. Hybrid-enzyme approaches, by taking advantage of the vast array of enzymatic properties that nature has evolved, as well as the strategies that nature has used to evolve them, are becoming an increasingly important avenue for obtaining novel enzymes with desired activities and properties.

Enzymes↗
PubMed1996

Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds.

Eukaryotic protein disulfide isomerase (PDI) is a 55-kDa enzyme with cysteine oxidoreductase, chaperone, and antichaperone activities that catalyzes disulfide formation and rearrangement in the eukaryotic endoplasmic reticulum. In Gram-negative bacteria, the formation of disulfide bonds in the periplasm is mediated by DsbA, a strong cysteine oxidase but an inefficient catalyst of disulfide bond isomerization with no known chaperone activity. We show that rat PDI (rPDI) secreted in the periplasmic space of Escherichia coli can catalyze the formation of disulfide bonds and complement several of the phenotypes of dsbA mutants. The function of rPDI was dependent on the dsbB gene, suggesting that the reoxidation of this eukaryotic enzyme involves direct interactions with bacterial redox proteins. Co-expression of rPDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) severalfold, an effect that was enhanced when reduced glutathione was added to the growth medium. Whereas PDI is thought to function primarily as an isomerase in the eukaryotic endoplasmic reticulum, rPDI failed to decrease the accumulation of two-disulfide folding intermediates of BPTI and thus did not appear to appreciably catalyze the rate-limiting step in the oxidative folding pathway of BPTI. These results demonstrate that expression of eukaryotic foldases in E. coli can be exploited to better understand their function in vivo and also to increase the yield of biotechnologically valuable proteins.

Animals↗
PubMed1994

The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm.

PreOmpA-bovine pancreatic trypsin inhibitor (BPTI) (Goldenberg, D. P. (1988) Biochemistry 27, 2481-2489) was expressed in Escherichia coli, and the folding pathway of the mature protein in the periplasmic space was analyzed by pulse-chase experiments. Folding intermediates were trapped with iodoacetamide, immunoprecipitated with antisera specific for either the reduced or the native protein, and resolved by electrophoresis. In vivo, native BPTI formed with a half-life of 7 min which is 3-fold faster than the optimal in vitro folding rate in growth media supplemented with low molecular weight disulfides. The measured in vivo half-life includes the time required for translocation and processing by leader peptidase and therefore represents the lower limit for the actual folding rate in the cell. In addition to the native species, two-disulfide intermediates accumulated in the cell at appreciable levels and did not chase to the native species for at least 20 min. We found that the folding of BPTI in E. coli was absolutely dependent on DsbA, a protein which accelerates the formation of disulfide bonds in the periplasm. In a dsbA mutant strain, trace amounts of oxidized BPTI could be detected only in cultures grown under strongly oxidizing conditions. In wild-type cells, the addition of different concentrations of GSH/GSSG or oxidized dithiothreitol did not affect the kinetics of BPTI oxidation by DsbA. Finally, even though the folding of BPTI in vitro decreases by almost 10-fold/unit pH decrease, folding in cells grown at pH 6.0 was only marginally slower than folding in cells grown at neutral pH, despite the fact that the pH of the periplasmic space varies in response to the extracellular fluid.

Animals↗
PubMed1994

Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.

The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.

Anilino Naphthalenesulfonates↗
PubMed1991

[The rating box--a new instrument for ambulatory detection of subjective variables].

For the acquisition of subjective variables in outpatients and inpatients, a new device--the Rating Box--has been designed. The Rating Box provides a standardized, date- and time-logged documentation of symptoms by the patient and rapid transfer of data to a personal computer. The simultaneous acquisition of several symptoms using different types of scales is possible. The acoustic alarm of the Rating Box reminds the patient to enter data at preselected times. The device is easy to handle, even by older patients, and has been successfully tested in the acquisition of pain variables in patients with a variety of painful conditions.

Data Collection↗