Browsing by Author "Ölschläger, Peter"

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Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression
(2003) Ölschläger, Peter; Lange, Stefan; Schmitt, Jutta; Siemann-Herzberg, Martin; Reuss, Matthias; Schmid, Rolf D.
In order to produce the atrazine-specific scFv K411B, it was expressed in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the scFv was N-terminally fused to the pelB leader, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly: The expression level of the scFv with leader was 2.3 times higher than that of the protein without leader. To further investigate this, the respective translation profiles were generated by coupled in vitro transcription/translation assays and gave according results. Periplasmic expression resulted in only 10% correctly folded scFv. The same percentage was obtained when the scFv was expressed in vitro, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of the scFv with EGFP fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.
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Impact of remote mutations on metallo-beta-lactamase substrate specificity : implications for the evolution of antibiotic resistance
(2005) Ölschläger, Peter; Mayo, Stephen L.; Pleiss, Jürgen
Metallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP 1 from IMP 6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants, with IMP-1 being the most active. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket and lead to enhanced activity. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion and increased sensitivity to mutations. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.
Unknown
In vitro and in silico models for in vivo events
(2002) Ölschläger, Peter; Schmid, Rolf D. (Prof. Dr.)
In Part A the atrazine-specific scFv (single-chain variable antibody fragment) K411B, that can be applied in herbicide analysis, was expressed either in the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the scFv was N-terminally fused to the pelB leader, whereas the unfused variant resulted in cytoplasmic expression. The expression level differed significantly: the extent of protein accumulation of the scFv with leader was 2.3 times higher than that of the protein without leader. To further investigate this, the respective translation profiles were generated by coupled in vitro transcription/translation assays and gave according results. Periplasmic expression resulted in only 10% correctly folded scFv. The same percentage was obtained when the scFv was expressed in vitro, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. Enhanced green fluorescent protein (EGFP) was fused C-terminally to the scFv to allow online monitoring during the production process. The resulting fusion protein could be used in a novel fluorescence immunoassay referred to as fluorophor-linked immunosorbent assay (FLISA) in order to measure functionality of the fusion protein. The scFv domain, which binds to the immobilized hapten, can be detected by measuring the fluorescence of the EGFP domain. The time-consuming binding of secondary antibodies and enzyme reaction, necessary for ELISAs (enzyme-linked immunosorbent assays), is not required. Consequently, the assay time of 1.5 hours needed to complete the FLISA is much shorter than that of comparable ELISAs, which require about 5 hours. The amount of the soluble fraction of cell extracts from Escherichia coli expressing the fusion protein and the normalized fluorescence signal showed a linear correlation with R2 > 0.99. The FLISA was used to determine the amount of functional scFv-EGFP fusion protein expressed in E. coli. The fusion protein could not be expressed in the periplasm but in the cytoplasm and showed a similar expression profile as the scFv. In vitro, the expression with and without the pelB leader rendered the same production profile as for the scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein. In a high-cell-densitiy cultivation, 25 gramms (2 gramms per liter) of scFv were produced. As the major fraction of recombinant protein was insoluble, a refolding method was developed yielding active scFv in nearly quantitative yields. Alternatively, active scFv could be purified from the culture supernatant to homogeneity by immobilized metal affinity chromatography. When applied to s-triazine analysis, the FLISA performed with scFv-EGFP fusion protein showed better results than the respective ELISA using scFv. In Part B an assay based on molecular dynamics simulations for the prediction of substrate specificities of the IMP-1 metallo-b-lactamase and variants towards cephalosporin antibiotics was developed. To establish a reliable modeling method for the active site, containing two zinc atoms, a purely nonbonded approach and a cationic dummy atom approach were tested. The latter was shown to give a better representation of the protein. The enzyme in complex with a mercaptocarboxylate inhibitor as it had been crystallyzed could be simulated well with this procedure at 300 K. The active site, i.e. the coordination of the two zinc atoms remained stable. Subsequently, the model was successfully extended to the free enzyme and to the enzyme in complex with a b-lactam bound as an intermediate. In this structure the b-lactam is already hydrolyzed and coordinated to one zinc via the carboxylate, to the other zinc via the anionic nitrogen resulting from the amide bond cleavage. Cephalothin, a substrate that is known to be converted well, remained stable in the intermediate structure at 300 K for 1.2 ns. The zinc-zinc distances in the free enzyme and the enzyme in complex with the b-lactam intermediate differed significantly and suggest that upon catalytic conversion of the antibiotic a movement of the zinc atoms occurs. To investigate IMP-6, a mutant differing in one amino acid from IMP-1, and other b-lactam substrates, the molecules were docked into IMP-1 and IMP-6, respectively, according to cephalothin. For these enzyme/substrate combinations literature data (kcat/KM values) were available and these showed significant differences. As not all combinations remained stable in the intermediate structure at 300 K, molecular dynamics simulations were performed at 100 K. After several simulations had been carried out for each combination, three parameters proved to be correlated to the experimental data: 1. For combinations with very inefficient hydrolysis, the anionic nitrogen lost contact to the respective zinc, 2. a deformation of the angle between the anionic nitrogen, the dummy atom (a component of the cationic dummy atom approach) and the zinc atom was observed for combinations with poor conversion and 3. an increase of the zinc-zinc distance occurred in inefficient enzyme/substrate combinations. The mentioned angle and distance were related in each combination and when they were plotted versus each other in a 2D chart, a grouping of inefficient, middle-rate and efficient metallo-b-lactamase/b-lactam combinations was observed.
Open Access
Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations
(2003) Ölschläger, Peter; Schmid, Rolf D.; Pleiss, Jürgen
Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc-containing IMP-1 metallo-beta-lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal-containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc-zinc distance increased by about 1 Å in the substrate complex compared to the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum-mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc-containing metallo-beta-lactamases as free proteins, in complex with inhibitors and in complex with substrates.