High Throughput Protein Expression And Purification Pdf
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- High-Throughput Protein Production and Purification
- High Throughput Protein Expression and Purification
- High-Throughput Protein Production and Purification
Metrics details. Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag His-tag or glutathione S-transferase GST -tag from bacterial expression systems. Proteins are expressed in well microplates and are purified by a fully automated procedure on a pipetting robot.
High-Throughput Protein Production and Purification
To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications.
We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms.
In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies. Proteins orchestrate the biological processes in living organisms through their interactions or manipulations of other biomolecules. Central to understanding the molecular function of a protein is to elucidate its three-dimensional 3D structure, which requires expression and purification of large quantities of highly pure and properly folded protein 1 , 2.
Yet, in the current proteomic era, the rapidly increasing demand for stable and functional proteins for research and commercial uses is still orders of magnitude higher than the available supply 3 , 4 , which largely hinders protein characterization, structure determination as well as the development of protein-based therapeutics.
The conventional approach to determining protein structures by X-ray crystallography relies heavily on the complicated and tedious screening of appropriate conditions for the growth of sufficiently large and well-ordered protein crystals, which emphasizes one of the major bottlenecks of structural biology.
Heterogeneously expressed proteins in the baculovirus-insect cell system can spontaneously form crystals within living cells, although this was commonly perceived as a somewhat rare event 5 , 6. Those crystals differ in many features including crystal morphology, stability, dimensions, growth dynamics, and subcellular localization 5.
The use of in vivo crystallography could eliminate the need for the extremely labor-intensive and time-consuming procedures associated with protein purification and in vitro crystallization. However, the number of protein structures available from in vivo-grown crystals has always been limited by their small size and their susceptibility to radiation damage 9 , 11 , These limitations have been recently overcome by the emergent technique of serial femtosecond crystallography SFX developed at X-ray free electron lasers XFELs as well as synchrotrons 10 , 11 , 12 , 13 , allowing data to be collected in a serial fashion from a stream of small nano- or micro-crystals for high-resolution structure determination 5 , 9 , 10 , This emerging concept of using serial crystallography with in vivo crystals opens new routes in structural biology of solving 3D protein structures 9 , 11 , and also highlights the significance of identifying novel in vivo crystal targets 12 , 13 , Thus, a high-throughput HT protein production pipeline built on the baculovirus-insect cell system will be extremely beneficial to the rapid screening for in vivo microcrystals that could be potentially advanced to serial crystallography for structure determination studies.
The baculovirus-mediated insect cell system has many advantages for protein expression—easy manipulation, low cost, accommodation of large DNA inserts, relatively high production level, and essential eukaryotic protein modifications similar to mammalian cells 15 , 16 , However, the procedures for inserting foreign genes into the baculoviral genome and repeated rounds of plaque purification necessary to isolate recombinants from the wild-type parental virus have been traditionally tedious, labor-intensive, and time-consuming 18 , 19 , which largely restricts its development for HT protein production The BacPAK6 system developed a triple-digested baculoviral genome that can force the homologous recombination with a transfer plasmid pBacPAK6 from TaKaRa to knock in the target gene and simultaneously restore the ORF , an essential viral gene that is partially deleted from this construct, so that only the recombinant viral genome can replicate in insect cells Although this strategy efficiently increases the percentage of the recombinants 17 , 24 , it still requires plaque purification and verification of the recombinant phenotypes following recombination.
In parallel with BacPAK6, the Bac-to-Bac system introduced the bacterial replicon and transposon attachment site to the baculoviral genome. This bacmid technology permits the propagation of the baculoviral genome in E. Additionally, a potential disadvantage of this system is the loss of target protein expression after serial passage of recombinant virus in insect cells 26 , and this might be associated with the genetic instability due to the presence of bacterial sequence retained in viral genome These limitations were resolved by the more recently developed flashBac system 28 , which represents a combination of bacmid technology and in vivo recombination with a transfer plasmid pOET from Oxford ET.
Upon homologous recombination, the target gene replaces the bacterial sequence that may cause poor genetic stability, simultaneously restoring ORF essential for replication. As no further separation techniques are required, the time and complexity of producing recombinant virus are remarkably reduced, thus making it suitable for automated HT protein expression 17 , The BacMagic system follows the same cloning principle and its latest bacmid has been further modified with deletions of several non-essential genes 29 , 30 , such as chitinase chiA , cathepsin v-cath , p10 , p74 , and p26 , which greatly improves the recombinant protein yield by reducing the protein degradation and increasing the recombinant biomass Given the above-mentioned advantages, the BacMagic-3 system along with the transfer plasmid pIEx Novagen were selected to build the protein production pipeline in this study.
However, the insertion of target gene into the pIEx vector requires restriction and ligation cloning that is not compatible with HT screening of many expression constructs. To reduce the time and resources needed to generate recombinant baculovirus at large scale, Radner et al. However, either multiple rounds of subcloning or substantial preparation of inserts from a genomic or cDNA template are required to obtain appropriately prepared PCR products prior to their insertion into the pIEx vector In the current study, we have developed a suite of Gateway-compatible variants of the pIEx vector such that any gene of interest in a Gateway donor clone can be transferred to these pIEx variants through one-step Gateway LR cloning to construct expression clones.
Workflow of the HT protein production and characterization pipeline. It enables: 1 easy selection of ORFs encoding target proteins from any existing Gateway clone libraries; 2 rapid and convenient HT construction of expression clones; 3 mass parallel expression screening of recombinant proteins with various tags; 4 affinity tag-based protein purification for functional characterization; and 5 fast and sensitive screening for in vivo microcrystal targets.
A suite of pIEx-based expression vectors for baculovirus-insect cell system have been generated to enable the rapid cloning of target genes into expression clones using Gateway technology. To construct a Gateway-compatible pIEx expression vector, the Gateway death cassette containing the ccdB lethal gene flanked by bacteriophage l site-specific recombination sites att R1 and att R2 was introduced downstream of the very late p10 promoter in the pIEx-cyto backbone Fig.
When mediated by LR clonase, target genes in frame from a Gateway donor clone replace the death cassette in pIEx expression vectors, resulting in pIEx expression clones Fig. Schematic drawing of pIEx expression vectors. Through In-Fusion cloning, the Gateway death cassette flanked by att R recombination sites was introduced downstream of the baculovirus very late promoter p10 and upstream of the ie1 terminator.
Sequences encoding fusion tags were inserted in the appropriate reading frame as indicated either upstream or downstream of the death cassette, which is later replaced by the gene of interest GOI upon Gateway LR reaction with the donor clone. A A gene of interest GOI in a donor clone is transferred to replace the death cassette containing the ccdB lethal gene in pIEx expression vector via Gateway recombination between att L and att R sites, producing a pIEx expression clone.
Thus, this pIEx vector collection provides a variety of options to tag target proteins depending on the desired downstream applications. Additionally, a tobacco etch virus TEV protease cleavage site was also introduced before or after the C-terminal or N-terminal tag so that, if desired, the fusion tags can be readily removed from the recombinant proteins with TEV protease Fig.
To demonstrate the mass parallel expression of recombinant proteins, a test collection of ORFs encoding 40 individual full-length proteins in Gateway donor clones were transferred into the pIEx expression vectors Supplementary Table S1. Additionally, these proteins are located in different subcellular compartments of their original organisms as well as involved in various biological processes. For proof-of-principle, selected proteins were tested in certain vectors Supplementary Table S2.
These results indicate that the protein production pipeline built on the suite of Gateway-compatible pIEx expression vectors can achieve rapid mass parallel cloning and expression of recombinant proteins with various fusion tags.
Successful parallel expression of target proteins. S3 , suggesting that the pipeline is capable of producing recombinant proteins stably and reproducibly. As examples to demonstrate the successful purification of recombinant proteins overexpressed in Sf9 cells using our new pIEx vectors, several proteins were isolated from the Sf9 cell cultures expressing His-tagged or GST-tagged proteins.
Protein purification. L lysate, UB unbound, W wash, E elution. Small-scale protein purification of cell extracts was performed for each of the pIEx expression vectors. The purified fraction was analyzed by Western blot using appropriate anti-fusion tag antibodies to confirm the identity of these targets. Out of 48 tested proteins, 24 were successfully purified and detected Supplementary Fig. To explore the feasibility of employing our tools in a HT structural biology pipeline using in vivo crystals, SONICC screening was performed to test for in vivo crystallization of recombinant proteins expressed using our pipeline.
Protein crystals in living insect cells are thereby detected by the green photons emitted. For amorphous precipitates or proteins in solution, the second harmonic signals cancel out. In contrast, no fluorescent particles nor crystals were detected in cells expressing only the EGFP tag Supplementary Fig.
To test whether the pipeline could identify any novel protein crystal targets, we expanded the SONICC detection to a large set of recombinant proteins for HT screening of in vivo microcrystals.
Thus, SONICC screening results may indicate the presence of crystalline particles grown in the living insect cells, which can be potentially used for further X-ray diffraction studies. High-throughput screening for in vivo crystals.
No signal was observed for proteins with cHalo tag or those without a tag. These results may represent the protein-dependent variations in the growth dynamics of in vivo microcrystals. The decline of the crystals follows the decrease in cell viability. Such differentiated fluorescence patterns might be associated with the difference in protein localization.
Time course of in vivo crystal formation. The presence of non-sharp particles was also observed in the rest of the samples, making the crystallinity of these targets less conclusive Supplementary Fig. Sf9 cells expressing EGFP alone were included as the negative control, and in contrast, no identifiable crystalline particles were seen in these cells with TEM Fig.
TEM of in vivo nanocrystals. The lower panels include the zoom-in view of the Sf9 cells shown in the upper panel for each target. Nano- or micro-crystals of different cellular localization, size, and morphology were observed for each target, as indicated by the red arrows.
EGFP was included as a negative control, and no identifiable crystals were observed inside the cells. Cell compartments cytosol, nucleus, and nuclear envelope NE have been highlighted for clarity in all images. Distinct morphology, size, and cellular localization were observed for each target Fig. Crystalline particles appeared hexagonal e. SGN1 structures. They were located in either cytosol e. It was also noticed that the nanocrystals for some targets were enclosed in cytoplasmic vesicles with either single e.
MLF2 or multiple e. The number of crystals per cells and the portion of cells containing crystals varied by targets. In this study, we have established a BEVS-based protein production pipeline to enable mass parallel recombinant protein expression and rapid screening for in vivo microcrystals. We first demonstrated the successful application of our new vectors for overexpression of proteins in Sf9 cells Fig.
S2 and their subsequent protein purification Fig. We further demonstrated that the new system can be used for in vivo crystallization screening prior to scaled-up production of microcrystals for structural and functional studies using a set of target proteins that vary in their host organism, molecular weight, and subcellular localization Fig.
Insect cells were chosen as the primary expression system for this pipeline because of their relatively high protein production level and broad eukaryotic protein processing abilities In vivo protein crystallization has been observed in nature in all kingdoms of life, where it is often functionally associated with protein storage, protection, and stabilization 5 , While native crystal formation usually provides advantageous functions for the organism, some accidental intracellular crystallization, induced by small environmental or structural changes, is considered to be related to certain diseases 5 , 6.
For example, Charcot-Leyden crystals CLCs 38 , 39 , which are spontaneously formed through auto-assembly of galectin protein in lymphocytes at diverse human tissues and body fluids, are involved in various allergic, parasitic, neoplastic and inflammatory disorders 40 , However, the role of CLCs in lymphocytes still remains obscure Other than the native crystal formation, the crystalline state has also been observed as a consequence of heterologous protein expression in host cells, which may be associated with the high local concentration of active protein.
Driven by this phenomenon, several additional examples of crystals has been discovered in living cells, including bacteria 42 , plant cells 43 , 44 , mammalian cells 45 , 46 , as well as baculovirus-infected insect cells 7 , The applicability of in vivo crystals in structural biology was first established at a synchrotron radiation source.
In , the first structure of a natively crystallizing protein, cypoviral polyhedra, was reported by Coulibaly and coworkers using the synchrotron diffraction approach
High Throughput Protein Expression and Purification
It seems that you're in Germany. We have a dedicated site for Germany. This book compiles key protocols instrumental to the study of high-throughput protein production and purification which have been refined and simplified over the years and are now ready to be transferred to any laboratory. Beginning with a section covering general procedures for high-throughput protein production, the volume continues with high-throughput protocols adapted to the production of specific protein families, as well as an extensive section on protocols combining high-throughput protein production and their micro-characterization. Written for the highly successful Methods in Molecular Biology series, chapters in this book include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, High-Throughput Protein Production and Purification: Methods and Protocols serves biochemists ranging from engineers, PhD students and post-doctoral fellows, to the heads of protein expression facilities and researchers, in pursuing this vital area of study. High-Throughput E.
Protocol DOI: The protocols outlined in this chapter allow for the small-scale test expression of a single or multiple proteins concurrently using several expression conditions to identify optimal strategies for producing soluble, stable proteins. The protocols. The protocols can be performed manually without the need for specialized equipment, or can be translated to robotic platforms. The high-throughput protocols begin with transformation in a well format, followed by small-scale test expression using auto-induction medium in a well format, finishing with purification in a well format.
Bacterial Expression Systems. Calmodulin Binding Peptide. Intein Mediated Purification. Each intein tag contains a chitin binding domain CBD for the affinity purification of the fusion protein on a chitin resin. Induction of on-column cleavage, using thiol reagents such as dithiothreitol DTT , releases the target protein from the intein tag.
High-Throughput Protein Production and Purification
Despite exciting advances in genome sequencing, isolating a protein from its expression system in its native form still presents a complex challenge. In High Throughput Protein Expression and Purification: Methods and Protocols , leading scientists detail the most successful protocols currently in use, including various high throughput cloning schemes, protein expression analysis, and production protocols. Cutting-edge and comprehensive, High Throughput Protein Expression and Purification: Methods and Protocols is an ideal reference for protein biochemists and all those who wish to apply these easy-to-use protocols to the many applicable fields. The volume is true handbook for protein biochemists, molecular biologists, biotechnologists, and other researchers and technicians who are involved in the many applicable fields.
It seems that you're in Germany. We have a dedicated site for Germany. Despite exciting advances in genome sequencing, isolating a protein from its expression system in its native form still presents a complex challenge. In High Throughput Protein Expression and Purification: Methods and Protocols , leading scientists detail the most successful protocols currently in use, including various high throughput cloning schemes, protein expression analysis, and production protocols.
Спокойствие, - потребовал Фонтейн. - На какие же параметры нацелен этот червь. На военную информацию. Тайные операции. Джабба покачал головой и бросил взгляд на Сьюзан, которая по-прежнему была где-то далеко, потом посмотрел в глаза директору. - Сэр, как вы знаете, всякий, кто хочет проникнуть в банк данных извне, должен пройти несколько уровней защиты.
Это наша главная цель. Простое число. Джабба посмотрел на таблицу, что стояла на мониторе, и всплеснул руками. - Здесь около сотни пунктов. Мы не можем вычесть их все одно из другого. - Многие пункты даны не в числовой форме, - подбодрила людей Сьюзан. - Их мы можем проигнорировать.
- В чем же чрезвычайность ситуации, из-за которой вы вытащили меня из ванной. Какое-то время Стратмор задумчиво нажимал на клавиши мышки, вмонтированной в столешницу письменного стола. После долгой паузы он наконец посмотрел ей в глаза и долго не отводил взгляда.
Они все, как один, - эгоцентристы и маньяки. Если им что нужно, то обязательно еще вчера. Каждый затраханный файл может спасти мир.
Несмотря на сомнения относительно быстродействия машины, в одном инженеры проявили единодушие: если все процессоры станут действовать параллельно, ТРАНСТЕКСТ будет очень мощным. Вопрос был лишь в том, насколько мощным.
- Он обесточен. - Вы оба настолько заврались, что в это даже трудно поверить. - Хейл сильнее сжал горло Сьюзан. - Если лифт обесточен, я отключу ТРАНСТЕКСТ и восстановлю подачу тока в лифт. - У дверцы лифта есть код, - злорадно сказала Сьюзан.
Недавно Стратмор сделал так, что Халохота снабдили новейшей игрушкой АНБ - компьютером Монокль. Себе Стратмор купил Скайпейджер, который запрограммировал на ту же частоту. Начиная с этого момента его связь с Халохотом стала не только мгновенной, но и абсолютно неотслеживаемой. Первое послание, которое он отправил Халохоту, не оставляло места сомнениям, тем более что они это уже обсуждали: убить Энсея Танкадо и захватить пароль.
Шифр до сих пор не взломан. Сьюзан от изумления застыла с открытым ртом. Она посмотрела на часы, потом на Стратмора. - Все еще не взломан.
Она взглянула на скоростное печатное устройство позади письменного стола шефа. В нем ничего не. - Сьюзан, - тихо сказал Стратмор, - с этим сначала будет трудно свыкнуться, но все же послушай меня хоть минутку.
Коммандер, вы ни в чем не виноваты! - воскликнула. - Если бы Танкадо был жив, мы могли бы заключить с ним сделку, и у нас был бы выбор.
Компьютер однократно пискнул. На экране высветилось: СЛЕДОПЫТ ОТПРАВЛЕН Теперь надо ждать. Сьюзан вздохнула. Она чувствовала себя виноватой из-за того, что так резко говорила с коммандером. Ведь если кто и может справиться с возникшей опасностью, да еще без посторонней помощи, так это Тревор Стратмор.
- Я шпионил за Стратмором. Эти письма в моем компьютере скопированы с терминала Стратмора - это сообщения, которые КОМИНТ выкрал у Танкадо. - Чепуха. Ты никогда не смог бы проникнуть в почту коммандера. - Ты ничего не понимаешь! - кричал Хейл.