Protein Crystallization Screening Research Articles

Droplet dynamics in asymmetric microfluidic junctions

Droplet-based microfluidics has received increasing interest over the last two decades, due to its extensive applications in drug delivery, cell encapsulation, protein crystallization, biochemical synthesis and high-throughput screening. As one of the key units in the microfluidic channels, asymmetric microfluidic junctions provide more opportunities to control and organize the droplets with different sizes, however, few studies have been reported to explore the effect of the junction angle on droplet behaviour in the asymmetric junctions. This work presents a numerical investigation of the droplet behaviour in asymmetric microfluidic junctions. The effect of the junction angle on the droplet breakup is explored in detail. Droplet breakup and non-breakup are found in asymmetric junctions. In the regime of the asymmetric breakup, the volume of the daughter droplet in the uphill branching channel is always larger than that in the downhill branching channel. Interestingly, in the regime of non-breakup, droplets are sorted into the uphill or downhill branching channels, suggesting that this asymmetric junction could be used for passive droplet sorting. The physical mechanisms behind droplet breakup and sorting are explored. Both the junction angle and the initial tip-to-tip length of the droplet play essential roles in the droplet behaviour in the asymmetric junctions. A phase diagram of the droplet behaviour is presented in this work. A scaling model is proposed to predict the transition between the breakup and non-breakup of the droplets in the asymmetric junctions.

Preparation and Characterization of Metal-Organic Framework Coatings for Improving Protein Crystallization Screening.

Modifying crystallization plates can significantly impact the success rate and quality of protein crystal growth, making it a helpful strategy in protein crystallography. However, appropriate methods for preparing nano-sized particles with a high specific surface area and strategies for applying these nanoparticles to form suitable coatings on crystallization plate surfaces still need to be clarified. Here, we utilized both an ultrasonic crusher and a high-pressure homogenizer to create a nano metal-organic framework (MOF), specifically HKUST-1, and introduced a solvent evaporation method for producing MOF coatings on 96-well crystallization plates to induce protein crystal growth. The morphology of MOF coatings on the resin surface of the plate well was characterized using optical and scanning electron microscopy. Compared to the control group, crystallization screening experiments on nine proteins confirmed the effectiveness of plates with MOF coatings. Applying MOF coatings to crystallization plates is an easy-to-use, time-efficient, and potent tool for initiating crystallization experiments.

Self-assembly in the transparent droplets formed during the screening of protein self-assembly conditions

Protein self-assemblies at the micro- and nano-scale are of great interest because of their morphological diversity and good biocompatibility. High-throughput screening of protein self-assembly at different scales and morphologies using protein crystallization screening conditions is an emerging method. When using this method to screen protein self-assembly conditions, some apparently transparent droplets are often observed, in which it is not clear whether self-assembly occurs. We explored the interaction between β-lactoglobulin and the protein crystallization kit Index™ C10 and observed the presence of micro- and nano-scale protein self-assemblies in the transparent droplets. The diverse morphology of the micro- and nano-scale self-assemblies in the transparent droplets formed by mixing different initial concentrations of β-lactoglobulin and Index™ C10 was further investigated by scanning electron microscope. Self-assembly process of fluorescence-labelled β-lactoglobulin was monitored continuously by laser confocal microscope, allowing real-time observation of the liquid-liquid phase separation phenomenon and the morphology of the final self-assemblies. The internal structure of the self-assemblies was gradually ordered over time by in-situ X-ray diffraction. This indicates that the self-assembly phenomenon within transparent droplets, observed in protein self-assembly condition screening experiments, is worthy of further in-depth exploration.

Depth-of-field extension in optical imaging for rapid crystal screening.

The depth of field (DoF) was extended 2.8-fold to achieve rapid crystal screening by retrofitting a custom-designed micro-retarder array (µRA) in the optical beam path of a nonlinear optical microscope. The merits of the proposed strategy for DoF enhancement were assessed in applications of second-harmonic generation imaging of protein crystals. It was found that DoF extension increased the number of crystals detected while simultaneously reducing the number of `z-slices' required for screening. Experimental measurements of the wavelength-dependence of the extended DoF were in excellent agreement with theoretical predictions. These results provide a simple and broadly applicable approach to increase the throughput of existing nonlinear optical imaging methods for protein crystal screening.

Searching for conditions of protein self-assembly by protein crystallization screening method

The self-assembly of biomacromolecules is an extremely important process. It is potentially useful in the fields of life science and materials science. To carry out the study on the self-assembly of proteins, it is necessary to find out the suitable self-assembly conditions, which have always been a challenging task in practice. Inspired by the screening technique in the field of protein crystallization, we proposed using the same screening technique for seeking suitable protein self-assembly conditions. Based on this consideration, we selected 5 proteins (β-lactoglobulin, hemoglobin, pepsin, lysozyme, α-chymotrypsinogen (II) A) together with 5 screening kits (IndexTM, BML, Morpheus, JCSG, PEG/Ion ScreenTM) to investigate the performance of these crystallization screening techniques in order to discover new optimized conditions of protein self-assembly. The screens were all kept at 293 K for certain days, and were analyzed using optical microscope, scanning electron microscope, transmission electron microscope, atomic force microscope, fluorescence microscope, and atomic absorption spectroscope. The results demonstrated that the method of protein crystallization screening can be successfully applied in the screening of self-assembly conditions. This method is fast, high throughput, and easily implemented in an automated system, with a low protein consumption feature. These results suggested that such strategy can be applied to finding new conditions or forms in routine research of protein self-assembly. KEY POINTS: • Protein crystallization screening method is successfully applied in the screening of self-assembly conditions. • This screening method can be applied on various kinds of proteins and possess a feature of low protein consumption. • This screening method is fast, high throughput, and easily implemented in an automated system.

Functionalization of the BCL6 BTB domain into a noncovalent crystallization chaperone.

The production of diffraction-quality protein crystals is challenging and often requires bespoke, time-consuming and expensive strategies. A system has been developed in which the BCL6 BTB domain acts as a crystallization chaperone and promiscuous assembly block that may form the basis for affinity-capture crystallography. The protein of interest is expressed with a C-terminal tag that interacts with the BTB domain, and co-crystallization leads to its incorporation within a BTB-domain lattice. This strategy was used to solve the structure of the SH3 domain of human nebulin, a structure previously solved by NMR, at 1.56 Å resolution. This approach is simple and effective, requiring only routine protein complexation and crystallization screening, and should be applicable to a range of proteins.

Schema Matching and Data Integration with Consistent Naming on Protein Crystallization Screens.

The data representation as well as naming conventions used in commercial screen files by different companies make the automated analysis of crystallization experiments difficult and time-consuming. In order to reduce the human effort required to deal with this problem, we present an approach for computationally matching elements of two schemas using linguistic schema matching methods and then transform the input screen format to another format with naming defined by the user. This approach is tested on a number of commercial screens from different companies and the results of the experiments showed an overall accuracy of 97 percent on schema matching which is significantly better than the other two matchers we tested. Our tool enables mapping a screen file in one format to another format preferred by the expert using their preferred chemical names.

Enhancing protein crystallization screening results using engineered nucleation features

Enhancing protein crystallization screening results using engineered nucleation features

Technology of high quality protein crystals’ obtaining aboard the ISS at execution of joint Japanese–Russian experiments

A series of space experiments aboard the International Space Station (ISS) associated with high-quality Protein Crystal Growth (PCG) in microgravity conditions can be considered as a unique and one of the best examples of fruitful collaboration between Japanese and Russian scientists and engineers in space, which includes also other ISS International Partners. X-ray diffraction is still the most powerful tool to determine the protein three dimensional structure necessary for Structure based drug design (SBDD). The major purpose of the experiment is to grow high quality protein crystals in microgravity for X-ray diffraction on Earth. Within one and a half decade, Japan and Russia have established an efficient process over PCG in space to support latest developments over drug design and structural biology. One of the keys for success of the experiment lies in how precisely pre-launch preparations are made. Japanese party provides flight equipment for crystallization and ensures the required environment to support the experiment aboard of the ISS’s Kibo module, and also mainly takes part of the experiment ground support such as protein sample characterization, purification, crystallization screening, and solution optimization for microgravity experiment. Russian party is responsible for integration of the flight items equipped with proteins and precipitants on board Russian transportation space vehicles (Soyuz or Progress), for delivery them at the ISS, transfer to Kibo module, and returning the experiments’ results back on Earth aboard Soyuz manned capsule. Due to close cooperation of the parties and solid organizational structure, samples can be launched at the ISS every half a year if the ground preparation goes smoothly. The samples are crystallized using counter diffusion method at 20 degree C for 1–2.5 months. After samples return, the crystals are carefully taken out from the capillary, and frozen for X-ray diffraction at SPring8 facility in Japan. Extensive support of researchers from both countries is also a part of this process. The paper analyses details of the PCG experiment scheme, unique and reliable technology of its execution, and contains examples of the application. Key words: International Space Station, Protein crystals, Microgravity, International collaboration.

Protein crystallization benefits from the rough well surface of a 48-well polystyrene microplate

Abstract It’s not clear whether the drop well surface can induce heterogeneous nucleation to increase the probability of screening success, although a medium-throughput 48-well SBS (the Society for Biomolecular Screening, USA) polystyrene microplate has been widely used for protein crystallization screening. Here, our accumulating data suggested that this 48-well SBS microplate with rough surface is beneficial for protein crystallization. It was observed that some protein crystals were only formed on the rough edges and/or walls of the drop well, but not on the flat bottom or in the crystallization drop. It was further demonstrated with two standard test proteins. The protuberances, grooves and cavities with varied sizes and shapes at micrometer-scale in the rough drop well wall can serve as nucleants and induce heterogeneous nucleation of protein crystals. The rough wall microplate provides a potent tool for protein crystallographers embarking on crystallization trials.

Protein crystallization screening using enhanced associative experimental design

Protein crystallization screening helps determine factors (e.g., salts, pH of buffers, ionic strengths, temperature, and type of precipitants) that are favorable for the formation of a large protein crystal suitable for X-ray crystallography. While existing commercial screens may not generate crystalline outcomes for difficult proteins, their outcomes could be used for recommending novel screens. Current methods for protein crystallization screening such as associative experimental design (AED) process only cocktails having one chemical per reagent while ignoring cocktails with multiple chemicals per reagent. To analyze cocktails having multiple chemicals per reagent, we propose enhanced associative experimental design (AED) that recommends novel crystallization conditions by analyzing the content of successful preliminary crystallization conditions. In wet lab experiments, our enhanced AED (AED$$^+$$) yielded ten new crystalline conditions for Tt189 (Nucleoside diphosphate kinase) in addition to 20 crystalline conditions generated by AED. Moreover, our AED+ allows pairing of crystalline or likely lead outcome with a non-crystalline outcome to generate novel crystalline conditions overcoming the limitation of AED requiring at least two good cocktails having at least one coming reagent.

Crystal Structure of Poly(Aspartic Acid) Hydrolase‐1

Poly(aspartic acid) (PAA) is a biodegradable synthetic polymer that is easily produced through heating aspartic acid followed by the subsequent addition of sodium hydroxide. Polymer applications range from drug delivery and biomimetics to hygiene products. Partial biodegradation of PAA is accomplished using poly(aspartic acid) hydrolase‐1 (PAAH‐1) which selectively cleaves a specific cross‐link. E. coli expressions of recombinant PAAH‐1 showed the production of significant amounts of protein which could be purified using a single Ni‐NTA column. Analysis of the PAAH‐1 sequence using the Phyre2 Protein Fold Recognition Server resulted in a 33% similarity to a “putative” poly(3‐hydroxybutyrate) depolymerase belonging to a family of enzymes that hydrolyze carboxylic ester bonds; however, no known structure of PAAH‐1 has been reported. Protein crystallization screening at the Hauptman‐Woodward Research Institute resulted in numerous protein crystallization hits being identified. Crystallization experiments were optimized in‐house and the structure was determined at 2.45Å using molecular replacement. The progress of PAAH‐1 characterization and its structure will be discussed.Support or Funding InformationNSF‐IUSE Grant: DUE 1611988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of the thermal shift assay and its power to predict protein and virus stabilizing conditions

Protein thermal shift assay (TSA) has been extensively used in investigation of protein stabilization (for protein biopharmaceutics stabilization, protein crystallization studies or screening of recombinant proteins) and drug discovery (screening of ligands or inhibitors). This work aimed to analyze thermal shift assay results in comparison to protein polymerization (multimerization and aggregation) propensity and test the most stabilizing formulations for their stabilization effect on enveloped viruses. Influence of protein concentration, buffer pH and molarity was tested on three proteins (immunoglobulin G, ovalbumin, and albumin) and results showed that each of these factors has an impact on determined shift in protein melting point Tm, and the impact was similar for all three proteins. In case of ovalbumin, molecular dynamics simulations were performed with the goal to understanding molecular basis of protein’s thermal stability dependence on pH. Effect of three denaturing agents in a wide concentration range on Tm showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on Tm for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing potential using cell culture infectivity assay (CCID50) and results showed lack of correlation with TSA results. Only weak correlation of Tm shift and protein polymerization measured by SEC-HPLC was obtained, meaning that polymerization cannot be predicted from Tm shifts.

Molecular biology enzymes, kits, and reagents market worth $26.7 bn by 2025

In order to solve increasingly challenging protein structures with crystallography, crystallization reagents and screen formulations are regularly investigated. Here, we briefly describe 96-condition screens developed at the MRC Laboratory of Molecular Biology: the LMB sparse matrix screen, Pi incomplete factorial screens, the MORPHEUS grid screens and the ANGSTROM optimization screen. In this short review, we also discuss the difficulties and advantages associated with the development of protein crystallization screens.

3D-Printed High-Density Droplet Array Chip for Miniaturized Protein Crystallization Screening under Vapor Diffusion Mode.

Here we describe the combination of three-dimensional (3D) printed chip and automated microfluidic droplet-based screening techniques for achieving massively parallel, nanoliter-scale protein crystallization screening under vapor diffusion mode. We fabricated high-density microwell array chips for sitting-drop vapor diffusion crystallization utilizing the advantage of the 3D-printing technique in producing high-aspect-ratio chips. To overcome the obstacle of 3D-printed microchips in performing long-term reactions caused by their porousness and gas permeability properties in chip body, we developed a two-step postprocessing method, including paraffin filling and parylene coating, to achieve high sealability and stability. We also developed a simple method especially suitable for controlling the vapor diffusion speed of nanoliter-scale droplets by changing the layer thickness of covering oil. With the above methods, 84 tests of nanoliter-scale protein crystallization under vapor diffusion mode were successfully achieved in the 7 × 12 droplet array chip with a protein consumption of 10 nL for each test, which is 20-100 times lower than that in the conventional large-volume screening system. Such a nanoliter-scale vapor diffusion system was applied to two model proteins with commercial precipitants and displayed advantages over that under microbatch mode. It identified more crystallization conditions, especially for the protein samples with lower concentrations.

An investigation of the effects of varying pH on protein crystallization screening

Protein crystallization occurs when the solution conditions are suitable for nucleation and growth of the crystals. pH, as one of the most important parameters that governs the protein crystallization process, can affect the conformation, activity, electrostatic interactions and solubility of protein in the solution. Hence, manipulating the pH is an important path to success in crystallizing proteins. In this paper, we show that varying pH levels over the incubation time during the crystallization process can help increase the chance of obtaining protein crystals. The process of varying the pH can be considered as one way to automatically search for a suitable pH for successful crystallization. This discovery can guide us to develop a new screening strategy in which varying the pH against the incubation time is the major process used during the crystallization screening. Our screening experiments verified that varying the pH is indeed a promising method to achieve efficient crystallization screening of a protein.

An investigation on the effect of surface roughness of crystallization plate on protein crystallization

Surface treatment by oxidizing the crystallization plates can significantly promote protein crystallization and requires no change to routine screening protocols; therefore, it is potentially useful for practical protein crystallization screening. However, experiments have shown that the amount of oxidants and the treatment process need to be optimized to achieve effective results. Searching for the suitable amount of oxidants, temperature and processing time for surface treatment of the plate will increase the workload and decrease the efficiency of the screening process. To solve this problem, a series of trials to determine suitable surface treatment conditions were conducted. Based on these experiments, not only was the most suitable processing condition for the optimal protein crystallization screening hits discovered but also the relationship between the treatment process and the protein crystallization screening hits was explored. With these results, the surface treatment of protein crystallization plates became easier and more effective, allowing the oxidizing surface treatment method to be applied on plates for use in routine protein crystallization screening.

Protein crystal screening and characterization for serial femtosecond nanocrystallography

The recent development of X-ray free electron lasers (XFELs) has spurred the development of serial femtosecond nanocrystallography (SFX) which, for the first time, is enabling structure retrieval from sub-micron protein crystals. Although there are already a growing number of structures published using SFX, the technology is still very new and presents a number of unique challenges as well as opportunities for structural biologists. One of the biggest barriers to the success of SFX experiments is the preparation and selection of suitable protein crystal samples. Here we outline a protocol for preparing and screening for suitable XFEL targets.

Protein Crystallization in an Actuated Microfluidic Nanowell Device.

Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies.

Optimizing Associative Experimental Design for Protein Crystallization Screening.

The goal of protein crystallization screening is the determination of the main factors of importance to crystallizing the protein under investigation. One of the major issues about determining these factors is that screening is often expanded to many hundreds or thousands of conditions to maximize combinatorial chemical space coverage for maximizing the chances of a successful (crystalline) outcome. In this paper, we propose an experimental design method called "Associative Experimental Design (AED)" and an optimization method includes eliminating prohibited combinations and prioritizing reagents based on AED analysis of results from protein crystallization experiments. AED generates candidate cocktails based on these initial screening results. These results are analyzed to determine those screening factors in chemical space that are most likely to lead to higher scoring outcomes, crystals. We have tested AED on three proteins derived from the hyperthermophile Thermococcus thioreducens, and we applied an optimization method to these proteins. Our AED method generated novel cocktails (count provided in parentheses) leading to crystals for three proteins as follows: Nucleoside diphosphate kinase (4), HAD superfamily hydrolase (2), Nucleoside kinase (1). After getting promising results, we have tested our optimization method on four different proteins. The AED method with optimization yielded 4, 3, and 20 crystalline conditions for holo Human Transferrin, archaeal exosome protein, and Nucleoside diphosphate kinase, respectively.