Towards protein crystallization as a process step in downstream processing of therapeutic antibodies: screening and optimization at microbatch scale

Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step.     

筛选蛋白质结晶条件的试剂盒研发进展

蛋白质结晶条件筛选是蛋白质分子三维结构解析的主要限速环节之一,因此发展筛选效率较高的结晶条件筛选试剂盒有着十分重要的意义.目前广泛应用的结晶条件筛选试剂盒的设计方法主要有不完全因子法、稀疏矩阵法和系统筛选法.结晶学家基于这三种方法研发了一系列用于蛋白质结晶条件筛选的试剂盒.评述了20世纪以来筛选试剂盒的设计方法及应用进展,并展望了其未来的发展方向.     

WaterLOGSY as a method for primary NMR screening: Practical aspects and range of applicability

WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.     

A deliberate approach to screening for initial crystallization conditions of biological macromolecules

A method to rationally predict crystallization conditions for a previously uncrystallized macromolecule has not yet been developed. One way around this problem is to determine initial crystallization conditions by casting a wide net, surveying a large number of chemical and physical conditions to locate crystallization leads. A facility that executes the rapid survey of crystallization lead conditions is described in detail. Results and guidelines for the initial screening of crystallization conditions, applicable to both manual and robotic setups, are discussed.     

Practical aspects of fumonisin production under laboratory conditions

Studies were performed to develop an efficient method for fumonisin toxin production in sufficient quantities for animal toxicological experiments, on the basis of three earlier published fumonisin toxin production methods. Three absolutely necessary factors were taken into account and tested in a serial experiment. TheFusarium verticillioides strain MRC 826 was directly inoculated onto soaked, autoclaved, whole maize kernels (50 g/1.71 jar). The inoculation was performed by standard spore suspension (l×l0⁶/ml), a 5/2 surface/volume culture was prepared and incubated at 25 °C for 5 weeks. To maintain the optimal a_w of approximately 1.00, the evaporated water was re-filled weekly. A final concentration of 4454±1060.9 ppm fumonisin B₁ was reached, with good repeatability. In the laboratory practice, consistent production of constant amounts of FB₁ can be obtained by applying the above settings.     

Development of a method for screening short-lived proteins using green fluorescent protein

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.     

High‐throughput protein refolding screening method using zeolite

We established a 96‐well‐plate‐based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96‐well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Advanced protein crystallization using water-soluble ionic liquids as crystallization additives

The application of five water-soluble, halogen-free, alkylammonium-based ionic liquids (ILs) as additives for advanced crystallization of lysozyme was investigated. Their biocompatibility was determined by long-term measurement of the overall mean relative enzyme activities. These were maximally reduced by about 10–15% when up to 200 g IL l⁻¹ was added. Sitting-drop vapor diffusion crystallization experiments revealed that the addition of some of the ILs led to less crystal polymorphism and precipitation was avoided reliably even at larger NaCl concentrations. The addition of ILs tended to result in larger crystals. The kinetics of lysozyme crystallization were significantly enhanced using ILs as crystallization additives, e.g. by a factor of 5.5 when 100 g ethanolammonium formate l⁻¹ was added. ILs with “soft” anions, such as formate or glycolate, were superior to ILs with “hard” anions, like nitrate.     

Selection of membrane protein targets for crystallization using PFO-PAGE electrophoresis

A method to rapidly assess the oligomeric composition of multimeric proteins is notably absent from reported schemes for high throughput production and crystallization of membrane proteins. In this report we have investigated the suitability of PFO-PAGE electrophoresis for this purpose and present examples where it proves highly informative in selecting conditions favouring the functional oligomeric state of the target protein. Features such as the ability to analyze several samples in parallel, including crude membrane extracts, suggest it will be highly adaptable to high throughput analysis of membrane proteins.     

Selection of membrane protein targets for crystallization using PFO-PAGE electrophoresis

A method to rapidly assess the oligomeric composition of multimeric proteins is notably absent from reported schemes for high throughput production and crystallization of membrane proteins. In this report we have investigated the suitability of PFO-PAGE electrophoresis for this purpose and present examples where it proves highly informative in selecting conditions favouring the functional oligomeric state of the target protein. Features such as the ability to analyze several samples in parallel, including crude membrane extracts, suggest it will be highly adaptable to high throughput analysis of membrane proteins.     

Practical aspects of screening of anabolic steroids in doping control with particular accent to nortestosterone radioimmunoassay using mixed antisera

A radioimmunoassay of two main classes of anabolic steroids, suitable for screening of these drugs in doping control is described and evaluated. Antisera against 17α-methyltestosterone-3-carboxymethyloxime-BSA were raised in both rabbits and goats. To enhance the specificity of nortestosterone assay two goat antisera against nortestosterone-17β-hemisuccinate-BSA (Ab. 1) and nortestosterone-3-carboxymethyloxime-BSA (Ab. 2) were raised, which recognized selectively 19-nor-3-oxosteroids and 19-nor-17β-hydroxysteroids, respectively. Using their mixture, the cross-reaction of naturally occurring steroids except for testosterone could be minimized. Tritiated steroids were used as tracers. Iodinated methyltestosterone and nortestosterone, prepared by simple melting of these steroids with Na¹²⁵I, could be used as alternative radioligands. Some questions concerning the expression of results and practical limitations of doping control of anabolics are discussed.     

Simple screening method for improving membrane protein thermostability

Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.     

Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases.

Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.     

High-throughput crystallization of membrane proteins using the lipidic bicelle method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent's inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization. Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. Bicelles have been successfully used to crystallize several membrane proteins (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer's arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.     

Attempts to rationalize protein crystallization using relative crystallizability

Protein crystal growth (PCG) remains the bottleneck of crystallography despite many decades of study. The nucleation zone in the two-dimensional-phase diagram has been used to evaluate the relative crystallizability of proteins, which is expressed as a percentage over the phase area delineated by experimental protein and precipitating agent concentration ranges. For protein-salts which are subject to a direct temperature effect on solubility, as represented by Egg Lysozyme, a decrease in temperature augments the nucleation zone percentage whereas for those with retrograde solubility as a function of temperature, for example fructose-1,6-bisphosphatase in the presence and absence of AMP, an increase in temperature can significantly enhance the relative crystallizability. These results have been confirmed by the number of "hits" using PEGs as precipitating agents in Sparse Matrix Screen experiments for different proteins and are in excellent agreement with the relative crystallizability. The relationship between solubility dependence, relative crystallizability and crystallization success, has been evidenced. Such crystallizability can become a guide to identify efficient crystallization regions, providing a rational approach to PCG and structural biology.     

Parallel production and verification of protein products using a novel high-throughput screening method

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.     

蛋白质自组装条件筛选形成的透明液滴中存在的自组装现象

微-纳尺度的蛋白质自组装体具有形貌多样性与良好的生物相容性,因而成为蛋白质自组装领域的研究热点。以蛋白质结晶条件的筛选手段高通量筛选不同类型蛋白质于不同尺度、不同形貌的自组装过程,是一种新兴的研究方法,具有重要研究意义。利用该方法进行蛋白质自组装条件筛选时,常会形成一些表观透明的液滴,其中是否有自组装现象的发生尚不明确。文中以β-乳球蛋白与蛋白质结晶试剂盒IndexTM C10相互作用为例进行探索,实验结果表明透明液滴中存在微-纳尺度的蛋白质自组装体。进一步通过扫描电镜观察不同初始浓度β-乳球蛋白与IndexTM C10混合形成的透明液滴中微-纳自组装体的形貌有所差别;通过激光共聚焦显微镜连续拍摄添加荧光标签的β-乳球蛋白形成自组装体的过程,可实时观察到液液相分离现象及最终形成的自组装体的形貌;通过原位X-射线衍射手段,可观察到自组装体内部结构随时间推移逐渐有序化的过程。以上研究表明,在以结晶条件筛选手段为基础的蛋白质自组装条件筛选实验中,透明液滴内的自组装现象具有深入探索的必要和价值。     

Theoretical aspects of genomic variation screening using DNA microarrays

We present a theoretical model for typical microarray-based single nucleotide polymorphism (SNP) assay of small genomic DNA amount. We derived the adsorption isotherm expressing the on-array hybridization efficiency in terms of genomic target sequence and concentration, oligonucleotide probe sequence and surface density, hybridization buffer, and temperature. This isotherm correctly describes the surface probe density effects, the sensitivity peak, and the melting temperature depression, and is in accord with published experiments. We discuss optimization of parallel SNP genotyping. Our estimates show that SNP detection at a single temperature in aqueous hybridization buffer is restricted by DNA regions that differ by less than 20% in GC content. We predict that the variety of genotyped SNPs could be substantially extended using an assay design with high probe density and a large fraction of probes hybridized.     

Selecting temperature for protein crystallization screens using the temperature dependence of the second virial coefficient

Protein crystals usually grow at a preferable temperature which is however not known for a new protein. This paper reports a new approach for determination of favorable crystallization temperature, which can be adopted to facilitate the crystallization screening process. By taking advantage of the correlation between the temperature dependence of the second virial coefficient (B(22)) and the solubility of protein, we measured the temperature dependence of B(22) to predict the temperature dependence of the solubility. Using information about solubility versus temperature, a preferred crystallization temperature can be proposed. If B(22) is a positive function of the temperature, a lower crystallization temperature is recommended; if B(22) shows opposite behavior with respect to the temperature, a higher crystallization temperature is preferred. Otherwise, any temperature in the tested range can be used.