Unnatural Protein Research Articles

Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System.

Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand.

Recent advances in cell-free unnatural protein synthesis

Cell-free unnatural protein synthesis (CFUPS) as an emerging approach for protein engineering research, has been successfully applied in basic scientific studies (e.g., protein-protein interaction, protein-nucleic acid interaction) and industrial production (e.g., pharmaceutical proteins, protein materials). CFUPS can improve the engineering freedom and the process control by allowing free addition of genetic elements and chemicals as research purposes. It also can give protein novel characteristics, structures and functions, including post-translational modification of proteins, incorporation of reaction handles, synthesis of biophysical probes and polymeric protein. This article systematically reviews the unnatural amino acid incorporation methods (global suppression, stop codon suppression, frameshift suppression and unnatural base-pairs) and the application advances of unnatural amino acids in protein modifications, biophysical probes, enzyme engineering, biomaterials and biopharmaceutical protein production. The opportunities and challenges of the CFUPS system development and the wide application of industrialization are also illustrated with details.

Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface.

HLA-B*27:05 is associated with the development of autoimmune spondyloarthropathies, but the precise causal relationship between the MHC haplotype and disease pathogenesis is yet to be elucidated. Studies focusing on the structure and cellular trafficking of HLA-B*27:05 implicate several links between the onset of inflammation and the unusual conformations of the molecule inside and at the surface of antigen presenting cells. Several lines of evidence emphasize the emergence of those unnatural protein conformations under conditions where peptide loading onto B*27:05 is impaired. To understand how cellular factors distinguish between poorly loaded molecules from the optimally loaded ones, we have investigated the intracellular transport, folding, and cell surface expression of this particular B27 subtype. Our findings show that B*27:05 is structurally unstable in the absence of peptide, and that an artificially introduced disulfide bond between residues 84 and 139 conferred enhanced conformational stability to the suboptimally loaded molecules. Empty or suboptimally loaded B*27:05 can escape intracellular retention and arrive at the cell surface leading to the appearance of increased number of β2m-free heavy chains. Our study reveals a general mechanism found in the early secretory pathways of murine and human cells that apply to the quality control of MHC class I molecules, and it highlights the allotype-specific structural features of HLA-B*27:05 that can be associated with aberrant antigen presentation and that might contribute to the etiology of disease.

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica.

Linkers for protein degradation.

The ability to subvert E3 ubiquitin ligases with small-molecule drugs offers tremendous promise for drug discovery. A new study demonstrates how structural and computational techniques can engineer and exploit unnatural protein–protein interfaces to design selective protein degraders.

Spin-Labeling of Proteins via Genetically Encoded Unnatural Amino Acids

Some of the major challenges of protein structure elucidation are proteins that do not crystallize well for x-ray studies and one that give overly convoluted NMR spectra. One way of resolving this issue is through the addition of radical spin-labels for study via electron paramagnetic resonance (EPR). This is usually done through disulfide linkages between a nitroxide spin-label and a cysteine residue of a protein, which allows for continuous wave and pulse EPR measurements. A wealth of information about the system is gained to provide structural insights. However, this requires one to remove native cysteines, which can significantly alter the overall structure and function of the protein. In this investigation, we use the unnatural amino acid, p-azido-phenylalanine, and an alkyne spin probe to “click” our probe to the model protein, T4 lysozyme. This ensures minimal structural perturbation, and allows this method to be used in proteins with native cysteines and disulfide bonds. Through our studies, we observed that this method works, but requires further refinement, and that it is also superior to the cysteine-based method due to the rigidity of the “click” bond, which gives more accurate measurements. Through further study, we hope to expand this method to more complex systems, and streamline a process for quick and easy unnatural protein labeling which will allow researchers to more easily study protein structure and dynamics.

Bacteria use expanded genetic code

The genome of every cell on Earth uses four DNA bases—adenine, thymine, cytosine, and guanine—to encode proteins. Chemists have long dreamed of expanding that set to create cells that work with both natural and unnatural nucleic acids. An expanded collection of bases could allow cells to synthesize unnatural amino acid-containing proteins that have new functions and could be useful as novel drugs, vaccines, and nanomaterials. But the closest researchers had gotten was to translate DNA with expanded base sets into proteins in test tubes. Now, Floyd E. Romesberg of Scripps Research Institute California and coworkers have gone all the way by putting an expanded genetic code into bacteria and getting the microbes to use it to synthesize an unnatural protein (Nature 2017, DOI: 10.1038/nature24659). The researchers got a strain of Escherichia coli to work with dNaM and dTPT3, aromatic DNA bases that pair with each other through complementary packing and

Targeted protein degradation: You can glue it too!

Proteolysis-targeting chimera (PROTACs) are synthetic molecules that recruit neo-substrate proteins to a ubiquitin ligase for ubiquitination and subsequent degradation. Structural insight into the VHL–MZ1–BRD4 complex reveals how the rationally designed MZ1–PROTAC molecule mediates degradation of an unnatural protein substrate.

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.

Biosynthesis and Directed Evolution of Unnatural Peptides and Proteins

Recently, the biosynthesis of peptides and proteins harboring chemical functionalities not found within the canonical 20 amino acids has been accomplished by harnessing natural post-translational modifications (PTMs) or by expanding the genetic repertoire with unnatural amino acids. These methods have vastly increased the chemical space that can be explored in directed evolution experiments, resulting in peptides and proteins with new or improved function. Although this area of synthetic biology is relatively new, it is rapidly emerging as a transformative research tool across multiple disciplines. Ultimately, the convergence of methodologies using PTM and expanded genetic repertoires will allow the biosynthesis of custom-tailored peptides and proteins with a rich variety of structures and functions. Keywords: directed evolution; post-translational modification (PTM); ribosomally synthesized post-translationally modified peptides (RiPPs); post-ribosomal peptide synthesis (PRPS); unnatural amino acids (uAAs); orthogonal aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs

Making sense of nonsense

Ryan Mehl is on a mission to make unnatural amino acids part of the natural order at the Unnatural Protein Facility.

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.

Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement

The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.

Observation of exclusively π-stacked heterodimer of indole and hexafluorobenzene in the gas phase

In this study, the structure of the indole···hexafluorobenzene dimer has been investigated in the gas phase by using resonant two photon ionzation (R2PI) and IR-UV double resonance spectroscopy combined with quantum chemistry calculations. We have confirmed the presence of exclusively π-stacked structure of the dimer from both experimental and theoretical IR spectra in the N-H stretching region. Observation of a single stable structure of the dimer has also been verified through 3D potential energy surface scan of the π-stacked dimer by varying the parallel displacement of the hexafluorobenzene unit simultaneously along the major and minor axes of the indole moiety. π-stacking interaction is present very often between the tryptophan and phenylalanine residues in proteins. But this interaction has not been observed earlier in the gas phase experiment by studying indole···benzene dimer because the N-H group of indole predominately directs towards the N-H···π hydrogen bonded T-shaped structure. The chosen molecular systems in this study not only rule out the possibility of the formation of the N-H···π bound T-shaped dimer but also enable the determination of the structure by probing the N-H group. The π-stacked indole···hexafluorobenzene dimer has a unique structure where the center of the hexafluorobenznene ring is aligned with the center of the shared bond of the indole ring. Our work provides useful insight in designing unnatural proteins having strong π-stacking interaction between the tryptophan and phenylalanine residues.

A foldamer-dendrimer conjugate neutralizes synaptotoxic β-amyloid oligomers.

Background and AimsUnnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimer's disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge.Methods and ResultsShort helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration.ConclusionsThe combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.

Determination and control of low‐level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system

Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross-reaction with a patient's endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX.

Bacterial Uptake of the Cell-Penetrating Peptide pVEC

The discovery of cell-penetrating peptides (CPPs), which can translocate through the cell membrane without the need for a receptor, has led to an increased interest in the design and development of peptide based drug delivery methods. CPPs are peptides of fewer than 30 residues derived from natural or unnatural proteins or chimeric sequences. The 18 amino acid long pVEC peptide is derived from murine vascular endothelial cadherin (VE-cadherin) protein, which mediates physical contact between adjacent cells by homophilic dimerization. The pVEC peptide has been shown to cross the cell membrane. The pVEC sequence, LLIILRRRIRKQAHAHSK, has a hydrophobic N-terminus and a positively charged midsection. The residues contributing to the uptake of pVEC across the membrane were previously examined by alanine mutagenesis studies (Elmquist, A., M. Hansen, et al., 2006, Biochimica et biophysica acta, 1758(6): 721-729) and the N-terminal residues were shown to be crucial in cellular uptake. Here, we use simulations to study the bacterial uptake of pVEC at atomic detail. Steered molecular dynamics simulations on the original pVEC and its various mutants are performed in an effort to determine the important peptide - membrane interactions on a residue basis and to elucidate the mechanism whereby pVEC passes through the membrane.

Protein Glycoengineering Enabled by the Versatile Synthesis of Aminooxy Glycans and the Genetically Encoded Aldehyde Tag

Homogeneously glycosylated proteins are important targets for fundamental research and for biopharmaceutical development. The use of unnatural protein–glycan linkages bearing structural similarity to their native counterparts can accelerate the synthesis of glycoengineered proteins. Here we report an approach toward generating homogeneously glycosylated proteins that involves chemical attachment of aminooxy glycans to recombinantly produced proteins via oxime linkages. We employed the recently introduced aldehyde tag method to obtain a recombinant protein with the aldehyde-bearing formylglycine residue at a specific site. Complex aminooxy glycans were synthesized using a new route that features N-pentenoyl hydroxamates as key intermediates that can be readily elaborated chemically and enzymatically. We demonstrated the method by constructing site-specifically glycosylated variants of the human growth hormone.

Importance of expression system in the production of unnatural recombinant proteins in Escherichia coli

In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while L-homopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system.

Design and engineering of an O2 transport protein

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. We introduce this method here with the first design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assemble a four helix bundle, position histidines to bis-his ligate hemes, and exploit helical rotation and glutamate burial on heme binding to introduce distal histidine strain and facilitate O2 binding. For stable oxygen binding without heme oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O2 affinities and exchange timescales match natural globins with distal histidines with the remarkable exception that O2 binds tighter than CO.