Small-scale, high-throughput method for plant N-glycan preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis

Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.     

A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification

Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein–ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.     

Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin

Elastin-like polypeptides (ELPs) undergo a reversible, soluble-to-insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature (T(t)). Incorporating a terminal ELP expression tag into the gene of a protein of interest allows ELP fusion proteins to be purified from cell lysate by cycles of environmentally triggered aggregation, separation from solution by centrifugation, and resolubilization in buffer. In this study, we examine the effect of ELP length on the expression and purification of a thioredoxin-ELP fusion protein and show that reducing the size of the ELP tag from 36 to 9 kDa increases the expression yield of thioredoxin by 4-fold, to a level comparable to that of free thioredoxin expressed without an ELP tag, while still allowing efficient purification. However, truncation of the ELP tag also results in a more complex transition behavior than is observed with larger tags. For both the 36 kDa and the 9 kDa ELP tag fused to thioredoxin, dynamic light scattering showed that large aggregates with hydrodynamic radii of approximately 2 microm form as the temperature is raised to above the T(t). These aggregates persist at all temperatures above the T(t) for the thioredoxin fusion with the 36 kDa ELP tag. With the 9 kDa tag, however, smaller particles with hydrodynamic radii of approximately 12 nm begin to form at the expense of the larger, micron-size aggregates as the temperature is further raised above the T(t). Because only large aggregates can be effectively retrieved by centrifugation, efficient purification of fusion proteins with short ELP tags requires selection of solution conditions that favor the formation of the micron-size aggregates. Despite this additional complexity, our results show that the ELP tag can be successfully truncated to enhance the yield of a target protein without compromising its purification.     

Expression and purification of an anti-Foot-and-mouth disease virus single chain variable antibody fragment in tobacco plants

Low-cost recombinant antibodies could provide a new strategy to control Foot-and-mouth disease virus (FMDV) outbreaks by passive immunization of susceptible animals. In this study, a single chain variable antibody fragment (scFv) recognizing FMDV coat protein VP1 was expressed in transgenic tobacco plants. To enhance the accumulation of scFv protein, the codon-usage of a murine hybridoma-derived scFv gene was adjusted to mimic highly expressed tobacco genes and fused to an elastin-like polypeptide (ELP) tag. This scFv–ELP fusion accumulated up to 0.8% of total soluble leaf protein in transgenic tobacco. To recover scFv–ELP protein from the leaf extract, a simple and scalable purification strategy was established. Purified scFv–ELP fusion was cleaved to separate the scFv portion. Finally, it was shown that the purified scFv proteins retained their capacity to bind the FMDV in the absence or presence of ELP fusion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineering a recyclable elastin‐like polypeptide capturing scaffold for non‐chromatographic protein purification

Previously, we reported a non‐chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin‐like polypeptide (ELP) to provide fast and cost‐effective protein purification. However, the bound dockerin‐intein tag cannot be completely dissociated from the ELP‐cohesin capturing scaffold due to the high binding affinity, resulting in a single‐use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium‐coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA‐mediated dissociation of the bound dockerin‐intein tag from the ELP‐cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non‐chromatographic based affinity method provides an attractive approach for efficient and cost‐effective protein purification. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:968–971, 2013     

Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage

While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale‐up have promoted the development of alternative non‐chromatographic separation methods. Here we developed a new non‐chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo‐responsive elastin‐like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin–dockerin pairs, which are high‐affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin‐tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self‐cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. Biotechnol. Bioeng. 2012; 109: 2829–2835. © 2012 Wiley Periodicals, Inc.     

High-throughput purification of recombinant proteins using self-cleaving intein tags

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.     

Purification of an elastin-like fusion protein by microfiltration

This article describes a simple and potentially scalable microfiltration method for purification of recombinant proteins. This method is based on the fact that when an elastin-like polypeptide (ELP) is fused to a target protein, the inverse phase transition behavior of the ELP tag is imparted to the fusion protein. Triggering the phase transition of a solution of the ELP fusion protein by an increase in temperature, or isothermally by an increase in salt concentration, results in the formation of micron-sized aggregates of the ELP fusion protein. In this article, it is shown that these aggregates are efficiently retained by a microfiltration membrane, while contaminating E. coli proteins passed through the membrane upon washing. Upon reversing the phase transition by flow of Milli-Q water, soluble, pure, and functionally active protein is eluted from the membrane. Proof-of principle of this approach was demonstrated by purifying a fusion of thioredoxin with ELP (Trx-ELP) with greater than 95% recovery of protein and with greater than 95% purity (as estimated from SDS-PAGE gels). The simplicity of this method is demonstrated for laboratory scale purification by purifying Trx-ELP from cell lysate using a syringe and a disposable microfiltration cartridge. The potential scalability of this purification as an automated, continuous industrial-scale process is also demonstrated using a continuous stirred cell equipped with a microfiltration membrane.     

Expression and purification of an antitumor-analgesic peptide from the venom of Mesobuthus martensii Karsch by small ubiquitin-related modifier fusion in Escherichia coli

To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the antitumor-analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO-AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni-NTA affinity chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N-terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Expression and purification of SARS coronavirus proteins using SUMO-fusions

Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.     

以类弹性蛋白多肽为标签的表达质粒构建及其用于木聚糖酶的非色谱纯化

【目的】旨在构建一个能以非色谱纯化目标蛋白的表达质粒,使用自行设计的类弹性蛋白多肽(ELPs)作为非色谱纯化标签,以纯化目标蛋白。该ELPs长度短,对盐非常敏感。【方法】从头设计了木聚糖酶,将其通过一段无规则卷曲同ELPs相连,合成了编码上述序列的基因,并构建重组表达载体pET-22b-SoxB-M2-S-ELP,转化至大肠杆菌BLR(DE3)中诱导表达,采用可逆相变循环经高速离心纯化木聚糖酶,并考察纯酶的酶学性质。【结果】成功构建了表达载体并表达,在pH=7.0时0.5 mol/L碳酸钠可使ELPs的相变温度降至22℃。在上述条件下,对木聚糖酶进行了非色谱纯化,其纯化倍数为3.2,回收率为21.2%,纯度为64.3%。经测定,未连接ELPs的酶、粗酶及纯化酶学性质基本一致,其最适温度为60℃,最适pH为6.0,最适反应时间为30 min,粗酶70℃保温1 h相对酶活仍有50%,为嗜热木聚糖酶,与预期相符。【结论】ELPs作为非色谱纯化标签纯化重组木聚糖酶具有操作简单、易于放大、成本较低的优势,故所构建的重组质粒可望通用于分离多种重组蛋白,具有较广泛的用途。     

Purification of recombinant apolipoproteins A-I and A-IV and efficient affinity tag cleavage by tobacco etch virus protease

The expression of recombinant apolipoproteins provides experimental avenues that are not possible with plasma purified protein. The ability to specifically mutate residues or delete entire regions has proven to be a valuable tool for understanding the structure and function of apolipoproteins. A common feature of many recombinant systems is an affinity tag that allows for straightforward and high-yield purification of the target protein. A specific protease can then cleave the tag and yield the native recombinant protein. However, the application of this strategy to apolipoproteins has proven somewhat problematic because of the tendency for these highly flexible proteins to be nonspecifically cleaved at undesired sites within the native protein. Although systems have been developed using a variety of proteases, many suffer from low yield and, especially, the high cost of the enzyme.We developed a method that utilizes the tobacco etch virus protease to cleave a histidine-tag from apolipoproteins A-I and A-IV expressed in Escherichia coli. This protease can be easily and inexpensively expressed within most laboratories. We found that the protease efficiently cleaved the affinity tags from both apolipoproteins without nonspecific cleavage. All structural and functional measurements showed that the proteins were equivalent to native or previously characterized protein preparations. In addition to cost-effectiveness, advantages of the tobacco etch virus protease include a short cleavage time, low reaction temperature, and easy removal using the protease''s own histidine-tag.     

Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

A self‐cleaving elastin‐like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self‐cleaving ELP‐intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP‐intein purification. Thus, this simple ELP‐based method should be applicable for the nonchromatographic purification of other recombinant, in vivo‐assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large‐scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.     

Optimization of ELP-intein mediated protein purification by salt substitution

In this paper we discuss improvements to our previously reported ELP-intein purification system described by Banki et al. [M.R. Banki, L. Feng, D.W. Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tags, Nat. Methods 2 (2005) 659–661; W.Y. Wu, C. Mee, F. Califano, R. Banki, D.W. Wood, Recombinant protein purification by self-cleaving aggregation tag, Nat. Protoc. 1 (2006) 2257–2262]. This method is based on the selective and reversible precipitation of ELP-tagged target proteins by gentle heating in the presence of high concentrations of sodium chloride. A critical aspect of this system is that the ELP tag is induced to self-cleave by a mild pH shift after purification. An examination of the Hofmeister series of ions suggested that salts other than sodium chloride may be more efficient for ELP precipitation. Specifically, by replacing sodium chloride with ammonium sulfate to induce ELP aggregation, we were able to reduce the required salt concentration by almost 4-fold, and the precipitation steps could be conducted at room temperature instead of 37 °C. This results in a cheaper, gentler, and more scaleable purification method. To demonstrate these advantages, green fluorescent protein and β-lactamase were purified using the newly optimized conditions in side-by-side comparisons to the previous method. The results indicate that both specific activity and yield were improved with the new conditions. These improvements thus significantly increase the attractiveness of this highly general and economical method for recombinant protein purification.     

Highly efficient and economical baculovirus expression system for preparing human papillomavirus type16 virus-like particle

To improve the existing human papillomavirus type16 (HPV16) virus-like particle (VLP) preparation, a highly efficient, economical and timesaving system was established. Sf-9 cells were infected with recombinant baculovirus containing the target gene encoding HPV16L1 protein with 6xHis tag, and harvested 72 h postinfection (p.i.) at 27 degrees. The ProBond(TM) purification system was used for protein purification. The molecular weight of expressed HPV16L1 protein was 58 kD as revealed by SDS-PAGE, and confirmed by Western blot. The purity of denatured and native HPVL1 proteins that were prepared were 91.9% and 71.5%, respectively, which corresponded to a yield of 2.26 mg denatured protein and 1.84 mg native protein per 2x10(7) cells. The proteins were further analyzed by mouse erythrocyte hemagglutination assay and hemagglutination inhibition assay, and there effects on VLP formation were also visualized by transmission electron microscopy. Results showed that the native protein purified was biologically active as natural HPVL1 protein, inducing the murine erythrocyte agglutination and VLP formation. In addition, the purified recombinant HPV16L1 native protein with 6xHis tag could self-assemble into virions in vitro. Hopefully, the present expression and purification system is promising to be convenient, timesaving and economical for preparation of HPV16 VLP vaccine.     

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.     

Challenges and recent advances in affinity purification of tag-free proteins

There is currently no generic, simple, low-cost method for affinity chromatographic purification of proteins in which the purified product is free of appended tags. Existing approaches for the purification of tagless proteins fall into two broad categories: (1) direct affinity-based capture of tag-free proteins that utilize affinity ligands specific to the target protein or class of target protein, and (2) removal of an appended affinity tag following tag-mediated protein capture. This paper reviews current state-of-the-art approaches for tagless protein purification in both categories, including specific examples of affinity ligands used for the capture of different classes of proteins and cleavage systems for affinity tag removal following chromatographic capture. A particular focus of this review is on recent developments in affinity tag removal systems utilizing split inteins.     

One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation

The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordinated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of [(VPGVG)(2)(VPGKG)(VPGVG)(2)](21) were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP bio- polymers. Biopolymers charged with Ni(2+) were able to interact with a His tag on the target proteins based on metal coordination chemistry. Purifications of two His-tagged enzymes, beta-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the utility of this general method and over 85% recovery was observed in both cases. The bound enzymes were easily released by addition of either EDTA or imidazole. The recovered ELPs were reused four times with no observable decrease in the purification performance.     

N‐terminal processing of affinity‐tagged recombinant proteins purified by IMAC procedures

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S‐transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine‐containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7‐triazacyclononane (tacn). The use of this tag‐tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli‐expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP‐1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI‐TOF MS analysis of the cleaved products from the DAP‐1 digestion of the recombinant N‐terminally tagged proteins confirmed the complete removal of the tag within 4‐12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn‐based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli‐expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.