pCold-GST vector: a novel cold-shock vector containing GST tag for soluble protein production

The production of recombinant protein in Escherichia coli is often hampered by low expression levels and low solubility. A variety of methodologies have been developed including protein production at low temperature, and fusion protein expression using soluble protein tags. Here, we present the novel cold-shock vector pCold-GST for high-level expression of soluble proteins in E. coli. This vector is a modified pCold I cold-shock vector that includes the glutathione S-transferase (GST) tag. The pCold-GST expression system developed was applied to 10 proteins that could not be expressed using conventional E. coli expression methodologies, and nine of these proteins were successfully obtained in the soluble fraction. The expression and purification of two unstable protein fragments were also demonstrated by employing a C-terminal hexa-histidine tag for purification purposes. The purified proteins were amenable to NMR analyses. These data suggest that the pCold-GST expression system can be utilized to improve the expression and purification of various proteins.     

A heme fusion tag for protein affinity purification and quantification

We report a novel affinity‐based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L ‐histidine‐immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme‐tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag‐HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.     

TRAF6-GST融合蛋白在大肠杆菌中的表达及纯化

目的:在大肠杆菌中表达肿瘤坏死因子受体相关因子6(TRAF6)与GST的融合蛋白并进行纯化。方法:采用PCR方法从肝文库中扩增编码TRAF6的DNA片段,将其插入原核表达载体pGEX-4T-2,构建GST-TRAF6原核表达载体,并转入大肠杆菌BL21(DE3)中,用IPTG诱导表达;用谷胱甘肽-琼脂糖珠亲和纯化表达的GST-TRAF6融合蛋白。结果:酶切鉴定和测序分析显示,长为1569 bp的TRAF6 DNA片段在pGEX-4T-2-TRAF6中的碱基序列、插入位点及读框正确,且位于表达载体的GST序列下游;经IPTG最佳浓度0.5 mmol/L诱导表达、亲和纯化后,获得了相对分子质量约85×103的GST-TRAF6融合蛋白。结论:构建了重组GST-TRAF6原核表达载体,获得了GST-TRAF6的大肠杆菌BL21表达菌株及GST-TRAF6融合蛋白,利于深入研究TRAF6的功能。     

Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag

In our work with designed minimalist proteins based on the bZIP motif, we have found our His-tagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6–C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1–4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more “open” protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.     

Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins

In this report, we describe a flexible, efficient and rapid protein purification strategy for the isolation and cleavage of glutathione-S-transferase (GST) fusion proteins. The purification and on-column cleavage strategy was developed to work for the purification of difficult proteins and for target proteins where efficient fusion-tag cleavage is essential for downstream processes, such as structural and functional studies. To test and demonstrate the flexibility of this method, seven diverse unrelated target proteins were assayed. A purification technique is described that can be applied to a wide range of both soluble and membrane inserted recombinant target proteins of differing function, structure and chemical nature. This strategy is performed in a single chromatographic step applying an on-column cleavage method, yielding "native" proteins in the 200 microg to 40 mg/l scale of 95-98% purity.     

Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.     

The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins.

A fusion tag, called FLAG and consisting of eight amino acids (AspTyrLysAspAspAspAspLys) including an enterokinase-cleavage site, was specifically designed for immunoaffinity chromatography. It allows elution under non-denaturing conditions [Bio/Technology, 6 (1988) 1204]. Several antibodies against this peptide have been developed. One antibody, denoted as M1, binds the peptide in the presence of bivalent metal cations, preferably Ca(+). Elution is effected by chelating agents. Another strategy is competitive elution with excess of free FLAG peptide. Antibodies M2 and M5 are applied in this procedure. Examples demonstrating the versatility, practicability and limitations of this technology are given.     

One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin

The 6xHis/Ni-NTA system allows rapid and efficient affinity purification of recombinant proteins from virtually any expression system. Protocols and tips for purification under both native and denaturing conditions are provided, as well as a rapid spin procedure for protein minipreps.     

A novel purification method for histidine-tagged proteins containing a thrombin cleavage site

A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified protein. The advantage of this method is that thrombin is used instead of imidazole in the final purification step. Imidazole can influence NMR experiments, competition studies, or crystallographic trials, and the presence of imidazole often results in protein aggregates. Removal of the His-tag results in a form of the protein of interest in which no additional tags are present, resembling the native form of the protein, with only three additional amino acids at the N-terminal side. Our method is compared with a more conventional method for the purification of the Azotobacter vinelandii NIFL PAS domain, overexpressed in Escherichia coli. It also proves to be successful for three different His-tagged proteins, the Klebsiella pneumoniae NTRC protein, and the A. vinelandii NIFA and NIFL proteins, and therefore it is a general method for the purification of His-tagged proteins.     

Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins

Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.     

Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris

The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines. We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest. We show that C-LYTA can be efficiently expressed and secreted in this host. Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices. It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes.     

Temporal regulation of VEID-7-amino-4-trifluoromethylcoumarin cleavage activity and caspase-6 correlates with organelle loss during lens development

Lens fiber cell differentiation involves extensive reconstruction of the cell's architecture, including the degradation and elimination of all membrane-bound organelles via a process that has been likened to apoptosis. Using caspase reporter assays under conditions in which nonspecific cleavage of the reporter peptides by the proteasome has been inhibited, we investigated whether any specific caspase activities are temporally correlated with this process of organelle loss. Extracts from neonatal mouse lenses contained strong VEID-7-amino-4-trifluoromethylcoumarin (AFC) and minor IETD-AFC and LEVD-AFC cleavage activities, but no DEVD-AFC cleavage activity. Further testing suggested that the VEID-AFC and IETD-AFC cleavage activities were likely due to the same enzyme. In lens extracts from rat embryos, VEID-AFC cleavage activity increased during the period when organelles are eliminated, between embryonic days 15.5 and 18.5, whereas procaspase-6 protein levels decreased, suggesting that this enzyme is responsible for VEID-AFC cleavage. By contrast, in extracts from alpha AE7 transgenic mouse lenses in which apoptosis was induced, strong DEVD-AFC cleavage activity and activated caspase-3 protein were detected. Thus, within the same tissue, different caspase activities can predominate depending on the context, normal differentiation versus apoptosis. These results highlight the difference between normal fiber cell differentiation and apoptosis and the capacity of the lens to differentially regulate these two processes.     

GST/ AEP 融合蛋白原核表达载体的构建、表达及鉴定

目的：为进一步研究抗癫痫肽（And—epilepsy peptide,AEP）的抗痫机制及筛选其相关作用蛋白,进行GST／AEP融合蛋白原核表达载体的构建及融合蛋白的表达。方法：通过PCR基因扩增对AEP基因进行扩增,并将其克隆于谷胱甘肽-S-转移酶（GST）融合蛋白表达质粒pGEX-4T-1中,经酶切、序列鉴定分析后,用该重组质粒转化大肠杆菌B121（DE3）,经IPTG诱导获得表达,并采用Western Blot进行检测。结果：成功构建了AEP原核表达载体,并在大肠杆菌B121中获得表达。结论：成功构建了GST／AEP原核表达载体,并表达了GST／AEP融合蛋白。