The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis

Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse® were well correlated with sonication. Two other methods, Bugbuster® and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

New Molecular Reporters for Rapid Protein Folding Assays

The GFP folding reporter assay [1] uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility [2]–[8], but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites [9]. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding [10]–[12]. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter [1] and the robustly-folding “superfolder” GFP [13]. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37°C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites

Experimental mapping of soluble protein domains using a hierarchical approach

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained

Performative Work: Bridging Performativity and Institutional Theory in the Responsible Investment Field

Callon’s performativity thesis has illuminated how economic theories and calculative devices shape markets, but has been challenged for its neglect of the organizational, institutional and political context. Our seven-year qualitative study of a large financial data company found that the company’s initial attempt to change the responsible investment field through a performative approach failed because of the constraints posed by field practices and organizational norms on the design of the calculative device. However, the company was subsequently able to put in place another form of performativity by attending to the normative and regulative associations of the device. We theorize this route to performativity by proposing the concept of performative work, which designates the necessary institutional work to enable translation and the subsequent adoption of the device. We conclude by considering the implications of performative work for the performativity and the institutional work literatures

Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control

Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar ‘superfolded’ proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues

Fluorescent Protein-Based Methods for On-Plate Screening of Gene Insertion

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening

Are individuals more risk and ambiguity averse in a group environment or alone? Results from an experimental study

Most decision-making research in economics focuses on individual decisions. Yet, we know, from psychological research in particular, that individual preferences can be sensitive to social pressures. In this paper, we study the impact of a group environment on individual preferences for risky (i.e., known probabilities) and ambiguous (i.e., unknown probabilities) prospects. In our experiment, each participant was invited to make a series of lottery-choice decisions in two different conditions. In the Alone condition, individuals made private choices, whereas in the Group condition, individuals belonged to a three-person group and group members' choices were aggregated according to either a majority or unanimity rule. This design allows us to study the impact of a group environment on individuals' attitude towards both risky and ambiguous prospects, while controlling for the decision rule used in the group. Our experimental results show that when individuals are in the Group condition, they tend to be less risk averse and more ambiguity averse than when they are not part of a group (Alone condition). Our experiment also suggests that the decision rule matters as it shows that these two trends tend to be stronger when the group implements a unanimity rule. Specifically, we found that individuals who belong to a group implementing a unanimity rule are significantly less risk averse than individuals who belong to a group that relies on the majority rule. We obtained a similar-but non-significant-result under ambiguity

Ataxin-1 Fusion Partners Alter PolyQ Lethality and Aggregation

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8±4.5% cell death versus 12.85±3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality