Intractable Proteins Research Articles

Considerations for safety assessment of intractable proteins expressed in genetically modified crops

Current requirements for safety assessment by regulatory agencies necessitate the production of multi-gram quantities of functionally active proteins newly expressed in genetically modified (GM) crops. This presents challenges for so-called intractable proteins (those that are difficult to isolate in an active form). Significant experience and knowledge have been gained by both GM crop developers and global regulatory agencies regarding the safety assessment of the newly expressed proteins (NEPs). By leveraging this knowledge and scientific data that have accumulated over the last several decades, the safety assessments of NEPs can be refined and improved. In this technical review, we examine the options for characterizing intractable NEPs and producing protein test substances from different sources to support the safety assessments of novel GM crops.

A Cell-Permeant Nanobody-Based Degrader That Induces Fetal Hemoglobin.

Proximity-based strategies to degrade proteins have enormous therapeutic potential in medicine, but the technologies are limited to proteins for which small molecule ligands exist. The identification of such ligands for therapeutically relevant but "undruggable" proteins remains challenging. Herein, we employed yeast surface display of synthetic nanobodies to identify a protein ligand selective for BCL11A, a critical repressor of fetal globin gene transcription. Fusion of the nanobody to a cell-permeant miniature protein and an E3 adaptor creates a degrader that depletes cellular BCL11A in differentiated primary erythroid precursor cells, thereby inducing the expression of fetal hemoglobin, a modifier of clinical severity of sickle cell disease and β-thalassemia. Our strategy provides a means of fetal hemoglobin induction through reversible, temporal modulation of BCL11A. Additionally, it establishes a new paradigm for the targeted degradation of previously intractable proteins.

Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.

Taking Aim at the Undruggable.

The term "undruggable" is used to describe a protein that is not pharmacologically capable of being targeted; recently, however, substantial efforts have been made to turn these proteins into "druggable" targets. Thus, "difficult to drug" or "yet to be drugged" are perhaps more appropriate terms. In cancer, a number of elusive targets fall into this category, including transcription factors such as STAT3, TP53, and MYC. Pharmacologically targeting these intractable proteins is now a key challenge of modern drug development, requiring innovation and the development of new technologies. In this article, we discuss some of the recent technologic and pharmacologic advances that have underpinned the erosion of the concept of undruggability. We describe recent successes in drugging the undruggable RAS (KRAS G12C and HRAS), and discuss the advances that have led to the validation of further targets previously believed to be undruggable, such as HIF-2α, BCL-2, MDM2, and MLL. Finally, we look to the future and describe important advances that are likely to have a major impact on targeting undruggable targets, such as the advent of proteolysis-targeting chimeras and protein-protein modulators, which are leading to considerable excitement surrounding the development of cancer targets.

Western Blotting of Membrane-Bound Proteins in Yarrowia lipolytica.

With the discovery of Western blotting as first described by Towbin et al. in 1979, the transfer and visualization of electrophoretically separated proteins on membranes has become the de facto method for the qualitative and quantitative detection of proteins of interest. In this method, proteins are resolved by electrophoresis on a polyacrylamide gel, followed by a transfer of the separated proteins onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane. Once immobilized on these membranes, the protein of interest can be detected and visualized by exploiting antigen-antibody interactions. However, not all proteins are amenable to easy detection by Western blotting. Integral membrane proteins are a class of proteins that are attached to a biological membrane through a series of transmembrane segments that span the width of the membrane. Due to the inherent hydrophobicity of these proteins and their tendency to aggregate, the characterization and detection of these proteins can be challenging. In this methods chapter, we present a protocol for the easy detection and quantification of these proteins in the industrially important oleaginous yeast Yarrowia lipolytica. The first protocol describes a Western blotting procedure to quantify soluble cytosolic proteins of interest in Yarrowia lipolytica from its total cell lysate. The second protocol describes modifications to the first that are done to enhance detection and quantification of membrane-bound proteins in Yarrowia lipolytica from its total cell lysate, without the need for isolating the membrane-bound proteins, for use in Western blotting. The immunoblotting strategies described here should serve as an efficient and simple guide to quantify both cytosolic and the intractable membrane-bound proteins in Yarrowia lipolytica.

Identification of additive formulations for enhanced shelf‐life and productivity of E. coli cell‐free protein synthesis extracts

Cell‐Free Protein Synthesis (CFPS) is a platform biotechnology that enables a breadth of applications, from expressing traditionally intractable proteins to metabolic engineering and point‐of‐care diagnostics. However, field applications have remained limited due to the poor shelf stability of aqueous E. coli cell‐free extracts, which contain a complex mixture of cellular machinery. Lyophilization of the E. coli extract improves shelf‐life, but remains insufficient for maintaining extract productivity over extended storage periods at room temperature. For many pure protein‐based biologics, a variety of additives have been identified that help to stabilize the protein for lyophilization, transportation, and storage. However, the advances made for pure proteins cannot be directly applied to the complex cell extract, and current investigations into improving cell extract stability via additives has been limited. Thus, we have addressed the cold chain limitation in cell‐free by mapping the chemical space of ten low‐cost additives with four distinct mechanisms of action in a combinatorial manner to elucidate their capacity to further stabilize lyophilized cell extract. We report three key findings: 1) unique additive formulations that maintain full productivity of the cell extract at 4°C and 23°C; 2) additive formulations that enhance extract productivity up to 195%; 3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. The identified additive formulations provide a simple and low‐cost advance toward making CFPS field‐ready.Support or Funding InformationBill and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF‐1708919)Comparison of traditional cellfree extract production and reaction setup with our modified workflow for improved stability and productivity to enable field applications.Figure 1

Improvements to Simplicity and Stability of the Cell‐Free Protein Synthesis Platform to Enable Applications in the Field and the Classroom

E. coli‐based cell‐free protein synthesis (CFPS) is a flexible platform technology for on‐demand protein production that allows users to express traditionally intractable proteins, perform high‐throughput screening, and, with augmentation, supports applications such as metabolic engineering and genetic code expansion. The broad utility of the CFPS platform arises from the elimination of the cellular membrane and capture of transcription and translation machinery in vitro. This obviates the need for living cells and creates an open system for direct manipulation of the environment of protein production. However, broad dissemination to field and classroom applications remains limited, as CFPS is dependent on technical scientific expertise for proper reaction setup and laboratory infrastructure for proper storage of reagents. Here, we report our efforts to improve broad accessibility of CFPS by 1) simplifying reaction setup and 2) improving shelf stability of the cell‐extract at various storage conditions. In order to simplify reaction setup, which currently requires precise pipetting of 10 reagents, we have combined the CFPS reagents into two stable premixes. These premixes can be stored at −20 °C for at least 4 months and allow the user to pipette fewer times with larger volumes for more accurate reaction setup by non‐experts. To improve the shelf stability of cell extract, which contains the sensitive transcription and translation machinery and is traditionally stored at −80 °C, we have characterized the functional effects of 10 protein stabilizing additives such as sugars, osmolytes, surfactants, and molecular crowding agents. Tests of single additives and combinations of two or three have allowed us to identify the landscape of small molecule additives that can support a variety of storage conditions ranging from −80 °C to room temperature to suit a variety of applications. When combined, these advances to reaction setup and cell extract composition provide the foundation for broad implementation of CFPS. Improved ease of reaction setup enables the use of CFPS by non‐experts, such as early career students, and improves efficiency for researchers and field applications. Additionally, increased storage stability diminishes reliance on the cold chain, making CFPS reagents easier to disseminate, commercialize, and use in the field. Together, these modifications provide a foundation for democratization of the CFPS platform.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An engineered E. coli ribosome with tunable translation rates enhances recombinant protein expression

Using E. coli for the expression of recombinant proteins in their soluble active form continues to be a challenge in biotechnology. Issues including low yields of full‐length, soluble, and functional proteins fundamentally limit discovery, development, and manufacturing of commercially, industrially, and medically relevant proteins. In many cases, improvements in protein solubility have been achieved by codon optimization, over‐expression of chaperone proteins, solubility tags, and by varying expression conditions. Optimization of these parameters is often done iteratively and on a case‐to‐case basis, which can be slow and expensive. To overcome limitations in recombinant protein expression, we report a genomically engineered strain of E. coli with a tunable ribosome enabling modulation in translation rates under isothermic conditions. Additionally, we show that cell extracts from this strain of E. coli can support a cell‐free protein synthesis platform capable of on‐demand production of recombinant proteins. This platform technology enables two key advances. First, we provide quantitative evidence that tuning protein synthesis rates improves soluble fractions of traditionally intractable proteins such as the human MEK1 and IRKβ. In each case, soluble fractions increased from <20% to >80% of total protein when compared with expression in BL21 cell extracts. 2) This platform technology is plug‐and‐play, allowing the user to identify the optimal translation rate condition for a desired protein in a high‐throughput manner, which can then be scaled‐up for desired protein yields. The timescale from DNA template to the optimized protein expression condition is 24–48 hours, notably faster than traditional methods involving cell transformations, and optimization of expression times and temperatures. Additionally, our data suggest that this approach obviates the need for solubility tags, enabling the expression of proteins in their wild‐type forms. Our work provides a new tool for studying protein folding in the context of ribosomal kinetics, and a new platform for manufacturing traditionally difficult proteins.Support or Funding InformationThis work was supported by DARPA and the the David and Lucille Packard Foundation Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Drugging the 'undruggable' cancer targets.

The term 'undruggable' was coined to describe proteins that could not be targeted pharmacologically. However, progress is being made to 'drug' many of these targets, and therefore more appropriate terms might be 'difficult to drug' or 'yet to be drugged'. Many desirable targets in cancer fall into this category, including the RAS and MYC oncogenes, and pharmacologically targeting these intractable proteins is now a key challenge in cancer research that requires innovation and the development of new technologies. In this Viewpoint article, we asked four scientists working in this field for their opinions on the most crucial advances, as well as the challenges and what the future holds for this important area of research.

Polarized monolayer cultures of human intestinal epithelial cell lines exposed to intractable proteins - In vitro hazard identification studies

• Intractable proteins did not produce adverse effects toward human intestinal epithelial cell (IEC) monolayers.

Constraining an Irregular Peptide Secondary Structure through Ring-Closing Alkyne Metathesis.

Macrocyclization can be used to constrain peptides in their bioactive conformations, thereby supporting target affinity and bioactivity. In particular, for the targeting of challenging protein–protein interactions, macrocyclic peptides have proven to be very useful. Available approaches focus on the stabilization of α‐helices, which limits their general applicability. Here we report for the first time on the use of ring‐closing alkyne metathesis for the stabilization of an irregular peptide secondary structure. A small library of alkyne‐crosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide. In addition, we report the crystal structure of the highest‐affinity derivative in a complex with its protein target 14‐3‐3ζ. It can be expected that the alkyne‐based macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins.

Single-Walled Carbon Nanotubes (SWCNTs) for Protein Engineering Applications

Protein engineering is a technique that has allowed researchers to overcome billions of years of evolution to create unnatural proteins that have been re-programmed to function and interact in ways unfounded in nature. It has been used to engineer proteins for a breadth of new purposes. This technique allows researchers to change the function of the protein by modifying its structure. Though a theoretical understanding of the relationship between the protein structure and function is helpful, unfortunately, the structure-function relation in the vast majority of proteins remains unknown. Directed evolution is a powerful protein engineering method that circumvents the need to define the structure-function relation of a protein. Briefly, after introducing random mutations to the protein, the collection of mutated proteins is screened to identify proteins with the new, desired function. The proteins with beneficial mutations are then isolated and mutated once more, and the process is repeated until the new protein function has been optimized. Scientists use this strategy to modify and/or improve protein properties, including stability, fluorescence, activity, and affinity to a specific target. One primary challenge in this method is developing a high-throughput screen for evaluating protein function. Researchers have established several high-throughput techniques to screen protein activities effectively using, for example, fluorescence-activated cell sorting or in vitro compartmentalization. However, these methods are largely limited to specific proteins and cannot be readily generalized. In our work, we use single-walled carbon nanotubes (SWCNTs) in a novel platform for investigating the functionality of a protein. SWCNTs emit fluorescence in the near-infrared spectral region between 900 nm and 1500 nm, a region that lies outside the optical window of most proteins and biomaterials in general. Further, SWCNTs demonstrate single-molecule sensitivities, with fluorescence emissions changing in response to perturbations in the charge, dielectric constant, and pH of the environment. We have exploited this sensitivity by interfacing SWCNTs with proteins that induce local environmental changes when active, thus allowing us to monitor protein activity by measuring the resulting changes to the SWCNT fluorescence. Combined with a moderate to high-throughput approach for acquiring SWCNT fluorescence, this platform provides biologists with a new tool for expediting the protein discovery process and engineering previously intractable proteins.

Hepatocellular carcinoma: targeting of oncogenic signaling networks in TRAIL resistant cancer cells.

Apoptotic response in hepatocellular carcinoma (HCC) cells is impaired because of interconnectivity of proteins into complexes and signaling networks that are highly divergent in time and space. TNF-related apoptosis-inducing ligand (TRAIL) has emerged as an attractive anticancer agent reported to selectively induce apoptosis in cancer cells. Although diametrically opposed roles of TRAIL are reported both as an inducer of apoptosis and regulator of metastasis, overwhelmingly accumulating experimental evidence highlighting apoptosis inducing activity of TRAIL is directing TRAIL into clinical trials. Insights from TRAIL mediated signaling in HCC research are catalyzing new lines of study that should not only explain molecular mechanisms of disease but also highlight emerging paradigms in restoration of TRAIL mediated apoptosis in resistant cancer cells. It is becoming progressively more understandable that phytochemicals derived from edible plants have shown potential in modelling their interactions with their target proteins. Rapidly accumulating in vitro and in-vivo evidence indicates that phytonutrients have anticancer activity in rodent models of hepatocellular carcinoma. In this review we bring to limelight how phytonutrients restore apoptosis in hepatocellular carcinoma cells by rebalancing pro-apoptotic and anti-apoptotic proteins. Evidence has started to emerge, that reveals how phytonutrients target pharmacologically intractable proteins to suppress cancer. Target-based small-molecule discovery has entered into the mainstream research in the pharmaceutical industry and a better comprehension of the genetics of patients will be essential for identification of responders and non-responders.

Characteristics and safety assessment of intractable proteins in genetically modified crops

Genetically modified (GM) crops may contain newly expressed proteins that are described as “intractable”. Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.

Crystallization of small proteins assisted by green fluorescent protein

The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties. Ubiquitin was accommodated in the lattice through interactions with the peripheral loops of GFP. However, in the GFP-UBM fusion crystal UBM formed extensive interactions with GFP and these interactions, together with UBM dimerization, mediated the crystal packing. Interestingly, the tyrosine residues that are involved in mediating crystal contacts in both GFP-ubiquitin and GFP-UBM crystals are arranged in a belt on the surface of the β-barrel structure of GFP. Therefore, it is likely that GFP can assist in the crystallization of small proteins and of protein domains in general.

Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag

We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.

Contribution of structural biology to clinically validated target proteins

We identified six groups of diseases expected to cause serious future health issues on the basis of a WHO report. Approved drugs for these diseases were associated with 409 target proteins; however, the percentage of selected proteins with full-length structural information deposited in the Protein Data Bank (PDB) was only 9.8%. The reason for the low percentage may be as a result of a disproportionate number of intractable proteins with multiple transmembrane regions and variable, or undefined glycosylation patterns, which impede protein preparation and crystallization, in such druggable proteins. We stress the importance of structural analysis of proteins, especially approved-drug target proteins, and the development of new methods to enable structural analyses of presently intractable proteins. In addition, we present an overview of large structural biology projects.

Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization

Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.

Cell engineering of Pseudoalteromonas haloplanktis TAC125: construction of a mutant strain with reduced exo-proteolytic activity

Background We have already shown that using cold-adapted bacteria as host vectors, some intractable proteins can be efficiently produced at temperature as low as 4°C [1,2]. Furthermore, we set up a cold gene-expression system implemented for the secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125). Such a system could effectively conjugate the positive effect of low temperature on the recombinant product solubility with the obvious advantages linked to extra-cellular protein targeting. This novel system makes use of the psychrophilic α-amylase from PhTAB23 [3] as secretion carrier. Several chimerical proteins were produced and used to test the versatility and efficiency of the novel secretion system. All the chimerical proteins were efficiently produced and secreted (Cusano AM, Ph. D thesis 2005 Universita di Napoli Federico II). However, bacteria belonging to Pseudoalteromonas genus are reported to secrete a wide range of exo-proteins, especially proteases. This feature could hamper both applicability and efficiency of the cold-adapted secretion system, due to the possible recombinant product degradation.

Yeast--a panacea for the structure-function analysis of membrane proteins?

In recent years, the scientific community has begun to realise that the structure-function analysis of membrane proteins has lagged considerably behind that of their soluble counterparts. A boom in the field of membrane protein biology has resulted in the tailoring of techniques for the cloning, expression, purification and characterisation of these somewhat intractable proteins and most notably in the optimisation of several alternative host systems for this purpose. This Review Article summarises the use of yeast as a host. Compared with other hosts, it is clear that yeast combines the advantages of eukaryotes with the ease of handling of prokaryotes. Moreover, this organism provides membrane protein biologists with a panacea for structure-function analyses, not least because the tools of yeast genetics are at their disposal.