Components of the E. coli envelope are affected by and can react to protein over-production in the cytoplasm.

BACKGROUND: Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway. In this context, we focused on the still poorly addressed relationship between protein production in the cytoplasm and the bacterial envelope, an active and reactive cell compartment that controls interactions with the environment and several major cellular processes. Results available to date show that the accumulation of foreign proteins in the cytoplasm induces changes in the membrane lipids and in the levels of mRNAs for some membrane proteins. However, a direct connection between membrane protein expression levels and soluble/aggregated protein accumulation in the cytoplasm has never been reported. RESULTS: By the use of a combined physiological and proteomic approach, we investigated the effects on the cell membrane of E. coli of the overexpression of two recombinant proteins, the B. cepacia lipase (BCL) and the green fluorescent protein (GFP). Both polypeptides are expressed in the cytoplasm at similar levels but GFP is fully soluble whereas inactive BCL accumulates in inclusion bodies.Growth and viability of the transformed cells were tested in the presence of different drugs. We found that chloramphenycol preferentially inhibited the strain over-producing GFP while SDS was more effective when BCL inclusion bodies accumulated in the cytoplasm. In contrast, both proteins induced a similar response in the membrane proteome, i.e. increased levels of LamB, OmpF, OmpA and TolC. Under all tested conditions, the lipopolysaccharide was not affected, suggesting that a specific rather than a generalized rearrangement of the envelope was induced. CONCLUSION: Taking together physiological and biochemical evidence, our work indicates that the E. coli envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Components of the E. coli envelope are affected by and can react to protein over-production in the cytoplasm

<p>Abstract</p> <p>Background</p> <p>Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway. In this context, we focused on the still poorly addressed relationship between protein production in the cytoplasm and the bacterial envelope, an active and reactive cell compartment that controls interactions with the environment and several major cellular processes. Results available to date show that the accumulation of foreign proteins in the cytoplasm induces changes in the membrane lipids and in the levels of mRNAs for some membrane proteins. However, a direct connection between membrane protein expression levels and soluble/aggregated protein accumulation in the cytoplasm has never been reported.</p> <p>Results</p> <p>By the use of a combined physiological and proteomic approach, we investigated the effects on the cell membrane of <it>E. coli </it>of the overexpression of two recombinant proteins, the <it>B. cepacia </it>lipase (BCL) and the green fluorescent protein (GFP). Both polypeptides are expressed in the cytoplasm at similar levels but GFP is fully soluble whereas inactive BCL accumulates in inclusion bodies.</p> <p>Growth and viability of the transformed cells were tested in the presence of different drugs. We found that chloramphenycol preferentially inhibited the strain over-producing GFP while SDS was more effective when BCL inclusion bodies accumulated in the cytoplasm. In contrast, both proteins induced a similar response in the membrane proteome, i.e. increased levels of LamB, OmpF, OmpA and TolC. Under all tested conditions, the lipopolysaccharide was not affected, suggesting that a specific rather than a generalized rearrangement of the envelope was induced.</p> <p>Conclusion</p> <p>Taking together physiological and biochemical evidence, our work indicates that the <it>E. coli </it>envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment.</p

Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.

In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.G.H. was supported by a CASE studentship from the Engineering and Physical Sciences Research Council and MedImmune and the Marie-Curie Research Training Network ENEFP. F.H. is a starting investigator of the European Research Council. Funding to pay the Open Access publication charges for this article was provided by the Engineering and Physical Sciences Research Council.This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/protein/gzv02

In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display)

A single mutation in the core domain of the lac repressor reduces leakiness.

BACKGROUND: The lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression. In Nature, the ability to detect and respond to environmental conditions comes at the cost of the encoded enzymes being constitutively expressed at low levels. The readily-switched regulation provided by LacI has resulted in its widespread use for protein overexpression, and its applications in molecular biology represent early examples of synthetic biology. However, the leakiness of LacI that is essential for the natural function of the lac operon leads to an increased energetic burden, and potentially toxicity, in heterologous protein production. RESULTS: Analysis of the features that confer promiscuity to the inducer-binding site of LacI identified tryptophan 220 as a target for saturation mutagenesis. We found that phenylalanine (similarly to tryptophan) affords a functional repressor that is still responsive to IPTG. Characterisation of the W220F mutant, LacIWF, by measuring the time dependence of GFP production at different IPTG concentrations and at various incubation temperatures showed a 10-fold reduction in leakiness and no decrease in GFP production. Cells harbouring a cytotoxic protein under regulatory control of LacIWF showed no decrease in viability in the early phases of cell growth. Changes in responsiveness to IPTG observed in vivo are supported by the thermal shift assay behaviour of purified LacIWF with IPTG and operator DNA. CONCLUSIONS: In LacI, long-range communications are responsible for the transmission of the signal from the inducer binding site to the DNA binding domain and our results are consistent with the involvement of position 220 in modulating these. The mutation of this single tryptophan residue to phenylalanine generated an enhanced repressor with a 10-fold decrease in leakiness. By minimising the energetic burden and cytotoxicity caused by leakiness, LacIWF constitutes a useful switch for protein overproduction and synthetic biology.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Scenario-Driven Search for Pedestrians aimed at Triggering Non-Reversible Systems

Abstract-This paper presents the results of an innovative approach to pedestrian detection for automotive applications in which a non-reversible system is used; therefore the aim is to reach a very low false detection rate, ideally zero, by searching for pedestrians in specific areas only. The great advantages of such an approach are that pedestrian recognition is performed on limited image areas-therefore boosting its timing performance- and no assessment on the danger level is finally required before providing the result to either the driver or an on-board computer for automatic manoeuvres. This system has been extensively tested on two prototype vehicles equipped with one laserscanner, one camera, and brakeby-wire technology both in Italy and Korea; this paper describes the extensive tests and shows performance measurements. I

Kinetics of aggregation and structural properties of proteins in inclusion bodies studied by Fourier transform infrared spectroscopy

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