We also explored expression of SEO proteins from Medicago truncatula , which are known to form large protein bodies, called forisomes [ 40 ]. Natural forisomes consist of several members of SEO protein family [ 34 , 35 ]. However, SEO proteins have not been analyzed at the individual protein level previously. TEM images of SEO1 proteins show that, at the initial stages of assembly, they form individual fibrils, which serve as building blocks for higher-order congregation Fig.
The obtained data are consistent with the functional roles of these proteins in vivo, where they participate in the molecular self-assembly of large 3D bodies. This is the first demonstration that such protein bodies can be generated in vitro from individual monomers. Due to significant instrumental advances in the electron microscopy such as the development of direct electron cameras and phase plates [ 42 , 43 ], cryo-electron microscopy cryo-EM holds a promise to be major imaging technology to study the structures of biological molecules in its native environment.
A double stacked hexameric ring geometry is evident. X-ray applications of cell-free-synthesized protein GS. The preparation of a wheat germ cell lysate is a tedious and a quite complex procedure [ 4 ]; therefore, the use of established commercial products is often recommended for obtaining reproducible results [ 28 ].
To minimize sample handling, the Promega WGCF kit offers a coupled setup where transcription and translation are run simultaneously. The CellFree Sciences WGCF kits have transcription and translation processes rather decoupled where each step is optimized individually to maximize the yield. If one needs to use an additive for the translation or change the reaction temperature, the latter setup offers a benefit of not interfering with the transcription stage. While the use of the specialized pEU vector is preferred to obtain higher yields of the given protein in this cell-free format, the most time-consuming steps are still the cloning of the respective gene, purification of the plasmid product, its subsequent sequencing and scale-up preparation.
While this is the final route to get high quantities of the desired protein product, it is always beneficial to know in advance if the protein is a good candidate to be expressed in this system and is worth the time invested. Fortunately, cell-free expression with somewhat reduced protein yields can be performed using PCR templates. The latter offers an ideal rapid screening to explore the protein potential to be expressed in a soluble and active form.
Based on our experiences in expressing a variety of proteins for different applications, we had a very high probability of producing good quality proteins from different organisms using this system. In combination with the wheat germ cell-free extract, this work presents a useful methodology for a multiscale pipeline covering all stages from cloning, screening potential candidates of protein synthesis to final production of micrograms quantities of a highly pure and tag-free protein.
The only exception was to use a His-tag-based purification for the 2D protein crystallography method which relies on a Ni-NTA functionalized lipid monolayer. Nevertheless, His-tag purification can be considered a good alternative strategy.
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In terms of yield, 0. While we show that such robotic platforms are not necessary, if available at a nearby institution, they could be used to replace the MEGA-scale expression using lower reaction volumes at potentially lower cost. The generated protein products are compatible with three major protein sample formats for high-resolution structural studies: 1 single-particle analysis, 2 2D crystallography and 3 3D macromolecular crystallography.
Obviously, the developed pipeline is applicable to any science area where interests lie in expressing of proteins in high yields and purities without the use of any specialized equipment and minimal time invested. Due to the fact that the cell-free expression platforms are gaining significant popularity, we believe that these comprehensive experiments will be useful for a wide range of scientific audience interested in exploring these systems especially as this platform can be replicated without major investments in instrumentation.
Gene sequences were either amplified from genomic DNA Ostreococcus tauri —cultured in-house, Medicago truncatula —gift from M. Knoblauch, or Arabidopsis thaliana —gift from H. To obtain genomic DNA from O. Genomic DNA was further ethanol precipitated and re-suspended in water.
The linearized pEU vector and the insertion fragment containing the desired gene were prepared by PCR. These fragments were further gel purified and quantified with NanoDrop c from ThermoFisher. Transformed E. For MIDI-scale plasmid-based protein expression 1. To prevent evaporation, wells were sealed with Parafilm.
To elute the bound protein, 0. Then, the supernatant fraction was collected, and the elution procedure was repeated one more time. For MAXI scale, all reagents were scaled linearly. The needed volume of gel suspension was first equilibrated in TBS buffer and then combined with the DTT-free translation mixture. Using the magnetic stand, the supernatant was removed and discarded, and the beads were then washed 3 times with TBS buffer 0.
Once the appropriate time was determined, the rest of the protein was cleaved in a similar fashion. Both lipids were purchased from Avanti Polar Lipids. The PELCO pad was then enclosed inside the sealed Petri dish, containing wet filter papers to create a humid environment and prevent evaporation. Negative staining of PDX1. For 2D protein crystals CCMK protein , an ultrathin carbon film on lacey carbon grid was not glow discharged. The carbon side was directly dropped down on top of lipid layer at the air—water interface of protein solution.
Each movie consisted of 16 frames encompassing total exposure of 0. All standard image processing from movie alignment to 3D map refinement was performed using CisTEM software [ 47 ] with default parameters apart from inputting the microscope Cs, image pixel size and estimated particle radii and symmetry. Crystals appeared over several days. JEE managed all aspects of this work. IVN performed genetics, molecular biology, cell-free expression and electron microscopy experiments.
RS assisted IVN with material handling. All authors read and approved the final manuscript.
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This research used resources of the Advanced Photon Source, a U. The plasmid constructs and all datasets, generated in this study, can be provided by the corresponding author upon reasonable request.
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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Skip to main content Skip to sections. Advertisement Hide. Download PDF. Protein structural biology using cell-free platform from wheat germ. Open Access. First Online: 10 November Background Systems biology seeks to understand genomic and proteomic changes between species or between individual cells to link compositional changes in the genome or proteome to the observed phenotype.
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From DNA to RNA: Transcription
PCR templates for cell-free expression can be prepared in short amount of time compared to construction of vectors and can, therefore, be used to quickly test target protein for expression and solubility. To incorporate those elements, a two-step PCR using a split primer design is suggested [ 30 , 31 ]. In addition, the Q5 enzyme demonstrates better capacity to produce long amplicons.
The main advantage of cell-free synthesis is the linear scalability of the reaction volumes where reactions can be run as MINI-expression trials to determine gene suitability or scaled up for high-resolution structural studies Fig. Naturally, the wheat germ extract has many endogenous proteins which will nonspecifically bind to nickel; therefore, the wheat germ extract optimized for His-tag purification was also employed. A single band of CCMK of desirable purity is clearly seen in the final fraction, which was later used for 2D crystallization trial discussed later.
Once we had an isolated protein in hand, the next step was to verify its activity, assembly state and identify whether it is a suitable candidate for high-resolution structural studies. Negatively stained CCMK proteins were clearly visualized as hexameric structures, mimicking the packing in the previous study [ 37 ].
Since protein synthesis consists of a series of standard biochemical reactions, such an approach is feasible in principle also for the process of mRNA translation. However, due to the cyclical nature of the elongation process and the fact that multiple elongation processes can occur on a single template, arriving at appropriate ODE structures is not as straightforward as for most other enzyme systems.
If an elongation step is to be more realistically presented as a series of reactions, rather than a single reaction, separate ODEs must be introduced for each reaction to be modelled. It is possible to accurately describe polysome structures with this approach by introducing different species for every possible combination of ribosome occupancies, thus for example denoting an mRNA containing two ribosomes on the fourth and tenth codons as R 4 R However, the number of ODEs required then approaches l-1 d , where d is the maximum density of ribosomes on the modelled mRNA.
Moreover, this type of model introduces a number of artefacts: for example, it inherently assumes that ribosome occupancy is not limited to one per codon, but can be multiple or fractions of one. In consequence, ribosomes in these types of models do not impede each other's progress when they collide. Despite the numbers of ODEs involved, Gerst and Levine [ 11 ] used a Laplace-transform approach to solve an ODE system for an RNA of 6 codons, which could accommodate up to two ribosomes at a time the actual size of ribosomes was unknown at the time. Considering the size of the equation system even for this simple case, it is clear why analytical solutions based on ODEs are rarely used.
However, the ideas from this paper were later modified to derive descriptions for the incorporation of radioactive label into newly synthesized protein, a widely used experimental technique [ 12 ]. ODE-based models also often form the basis for computer-based numerical analyses see below. As an alternative to the deterministic ODE-based models, approaches based on the statistical properties of ribosome movement were explored initially by Zimmerman and Simha [ 13 , 14 ], and then refined by MacDonald and Gibbs [ 15 , 16 ]. These groups considered mRNAs as lattices on which ribosomes move with specific hopping probabilities, the latter being functions of the intrinsic kinetics of elongation; the ratio of initiation, elongation and termination rates which determine the ribosome density on the message ; and the probability that progress of a ribosome is unimpeded by preceding ribosomes which is itself a function of the ribosome density on the message.
The statistical approach proved popular in a number of modelling studies which modified the basic solutions provided by MacDonald and Gibbs for the investigation of specific questions, such as competition between messenger RNAs with different initiation rate constants [ 17 — 20 ], genome-wide translation systems in E. Despite its origin in attempts to describe ribosome movement along mRNAs, TASEP -based approaches were initially not widely used to address protein synthesis problems. Instead, they enjoyed significant success in analyses of vehicular road traffic flows [ 24 ].
From there, they found their way back into biology, and the last ten years have seen an explosion of TASEP -based studies of translation [ 25 — 39 ]. Recent modifications to the basic TASEP allowed analyses of codon-specific elongation rates [ 26 — 28 , 30 , 33 ], limiting supplies of ribosomes [ 31 , 37 , 38 ] or tRNAs [ 35 ], and traffic on circularised mRNAs [ 25 ], thus making the approach more physiologically relevant. Several recent studies also described approaches that go beyond the description of ribosome movement as a simple hopping probability, and instead consider the detailed sub-steps of the translation elongation cycle [ 29 , 32 , 33 , 36 , 39 ].
In addition to the approaches described above, specialised approaches were developed to address specific questions on the process of translation. Other studies have used specialised mathematical models to analyse the effect of mRNA decay on polysome shape [ 41 — 44 ], to analyse the effect of highly expressed heterologous mRNAs on rare tRNA availability [ 45 ], to quantify differences in selective pressure between synonymous codons [ 46 ], to determine in how far the avoidance of nonsense errors contributes to natural selection between synonymous codons [ 47 ], and to quantify the effect of frame-shift errors on translation [ 48 ].
All of these models are largely based on statistical analyses of the behaviour of ribosomes on mRNAs. While analytical solutions can yield meaningful descriptions of the behaviour of ribosomes, the theory on which they are based is usually difficult for non-specialists. Computer simulations are generally used in two ways, both requiring knowledge of rate constants or rates for the individual reactions that form the model.
The first approach is to establish systems of ordinary or stochastic differential equations that describe every reaction of the modelled process. These ODE or SDE systems are then used to compute numerical approximations of the development of the system over time, for a given set of parameters and starting conditions.
Although this approach is sometimes used to model the actual movement of ribosomes on mRNAs [ 50 — 53 ], it suffers from the same difficulties as outlined above for analytical models of translation regarding the size of equation systems that can result from describing each possible codon:ribosome complex as an individual species. However, numerical approximations have been the main approach for modelling the sub-processes of translation initiation [ 54 — 57 ] and termination [ 58 ].
In this Monte Carlo approach, randomly generated numbers are used to a select one reaction from all the reactions possible in the system, and b decide whether or not this reaction will proceed, by comparing the random number to a probability value derived from the rate of the selected reaction. This approach has been used to simulate ribosome movement on mRNAs [ 49 , 59 — 66 ], as well as the stochastic tRNA sampling process underlying translation elongation [ 67 ].
The average results from many individual Monte Carlo simulations converge exactly on the average behaviour of the described system, but this approach is computationally expensive for large systems. A specific variant of Monte-Carlo simulations are so-called agent-based models [ 10 , 68 ].
In these types of model, the modelled particles such as ribosomes or mRNAs are represented by individual variables, rather than pools of particles as in non-agent-based approaches. It is noteworthy that the very first models of mRNA translation used this type of data structure, and thus constituted rudimentary agent-based models long before this term was in use [ 49 , 59 ].
Inherent assumptions arising from the formulation of these models included an unlimited supply of ribosomes and tRNAs, as well as a codon-independent rate of movement. The complete set of input parameters consisted typically of values for the three rates, the mRNA length, and the number of codons covered by one ribosome. From these beginnings, models grew with the biological knowledge on one hand, and with computational power on the other. This led to the development of more fine-grained models, in which one or more of the translational phases were assumed to proceed in multiple sub-steps Figure 3.
Thus, Godefroy-Colburn and Thach described initiation via five sub-reactions [ 20 ], Heyd and Drew dissected the elongation step into seven sub-reactions [ 51 ], and de Silva et al. All of these models analysed the full ribosome cycle. Even more fine-grained models were developed for translation initiation 12 reactions, [ 56 ] and elongation 17 reactions, [ 67 ] , although these were implemented as stand-alone models that did not consider the respective other two sub-processes.
A fine-grained model of one translation elongation step. The codon decoding reactions k 1 -k 7 , and translocation reactions k 8 -k 15 are shown with the known, biochemically distinguishable reaction steps compiled from refs. Shape changes indicate conformational changes in the translation elongation factors. The selective expansion and collapse of individual sections of translation into more or less fine-grained reaction systems has become a convenient strategy to consider as much detail as is required for any particular analysis, without incurring unnecessary computational cost by modelling everything in detail.
Throughout all of the studies cited above, the aims of computational and mathematical biologists have remained remarkably constant and centred around two important themes. To aid in the interpretation of experimental data. The very first modelling studies were conducted with the stated aim of exploring the relationship between polysome profiles and translational rate constants, because it was realised that the relationship between the two was complex, but that the establishment of any defined relationships between the two would be a useful tool for experimentalists.
To investigate rate-limitations in the process of translation. Although recent results indicate that the control of translation is highly distributed and the idea of a single rate-limiting step is thus likely to be an oversimplification [ 57 , 70 ], discussions on the role of individual translation factors as rate-limiting or not rate-limiting constitute an important part of the literature on translation [ 71 — 77 ].
Among the most successful findings from modelling studies, at least as judged by the numbers of citations received, was Lodish's prediction that message-specific translational control could be exerted by canonical translation factors [ 18 ].
It probably helped the success of this study that the author provided experimental evidence for the correctness of his model-derived predictions in the same paper as the model. Another study by Rapoport et al [ 23 ] analysed signal recognition particle SRP -mediated pausing during the translation of secreted proteins.
The principal findings from this study were that SRPs arrest ribosomes individually rather than arresting an entire polysome at a time , and that the translational arrest would only have functional consequences under conditions of strongly limiting SRP abundance. These findings were later confirmed experimentally, and remain widely cited.
Initial modelling studies focussed in particular on the question whether initiation or elongation activity limited protein production rates.
For individual mRNAs and physiological ratios of initiation to elongation rates, initiation appeared clearly limiting [ 16 ]. This view was strongly taken up by the experimental community, and many recent papers still contain general statements referring to translation initiation as the rate limiting step of translation.
A parameter that is particularly important in this context is the availability of free ribosomes. Formal control analyses showed that as the levels of available non-translating ribosomes approach zero, control over cell-wide translational activity is quantitatively transferred from initiation to elongation [ 19 ]. This is because under ribosome-limiting conditions, initiation events cannot occur unless translating ribosomes finish synthesizing the last protein and become available for initiation on the next message.
Under such conditions, faster or slower average translation elongation rates can significantly control rates of subsequent initiation events. It is interesting to note that all current computational models envisage that a ribosome which has finished translating an mRNA exchanges with the cytoplasmic ribosomal pool, and selection of the next mRNA to be translated by that ribosome occurs in a strictly stochastic manner. However, recent experimental results show that eukaryotic ribosomes may translate mRNAs in multiple cycles before entering the free ribosome pool [ 78 ].
This would affect the control of translation profoundly, and compared with single-cycle models, it could transfer significant levels of control to the elongation stage. At what level of ribosome depletion control is transferred to elongation depends in complex ways on the codon composition of the genome. The Advances in Protein Chemistry and Structural Biology series is an essential resource for protein chemists. Each volume brings forth new information about protocols and analysis of proteins, with each thematically organized volume guest edited by leading experts in a broad range of protein-related topics.
Researchers and specialists in protein structure and interactions, cancer biology, psychiatry and mass spectrometry. In Dr. Donev was appointed Senior Lecturer at Swansea University. He has published more than 60 research papers, chaired scientific meetings and given invited plenary talks. Rossen Donev has consulted on projects related to development of treatments for neurodevelopmental disorders and cancer therapies.
He serves as Editor-in-Chief of the Advances in Protein Chemistry and Structural Biology and on editorial board of several other journals. His research interests include signaling pathways involved in neuropsychiatric disorders and tumor escape from the immune system, and development of therapeutic strategies for their treatment.
Advances in Protein Chemistry and Structural Biology, Volume 101
More recently he has focused on design of antibody-based oral treatment of pathological conditions of the gastrointestinal tract. We are always looking for ways to improve customer experience on Elsevier. We would like to ask you for a moment of your time to fill in a short questionnaire, at the end of your visit.
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