Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and β-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.     

Purification of green fluorescent protein using a two-intein system

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.     

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.     

Iron Oxide Nanoparticle Assisted Purification and Mass Spectrometry Based Proteolytic Mapping of Intact CD4+T Cells from Human Blood

Abstract

Iron oxide nanoparticles have been used for many years as clinical applications. We have developed a rapid immunoaffinity isolation method of CD4⁺T cells from a mixed cell population of human blood using iron oxide nanoparticles. Anti CD4-antibody has been attached to iron oxide nanoparticles after its surface modification. The antibody tagged iron oxide nanoparticle beads are simply incubated with the mixed cell population of human blood and CD4⁺T cells are purified using an external magnetic field. The purification level was checked by fluorescence microscopy and flow cytometry. The purified CD4⁺T cells were digested with trypsin with different time periods and the products were analyzed by MALDI-TOF mass spectrometry, without further fractionation or purification, to obtain its proteome pattern. A database search showed a number of peptide masses matched specific to T-cell peptide masses. These results indicate that iron oxide nanoparticles are useful for CD4⁺T cell purification, and mass spectrometry based proteolytic fingerprint is simple and swift for identifying putative surface biomarkers from the whole cell surfaces.     

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.     

Expression, purification and characterization of a novel soluble human thymosin alpha1 concatemer exhibited a stronger stimulation on mice lymphocytes proliferation and higher anti-tumor activity

Thymosin alpha 1 (Tα1) has immunomodulatory and anti-tumor effects in patients and has been commercialized in worldwide. An innovative technique is therefore impending to achieve high-yield expression and purification of Tα1 to meet the increasing requirements for clinical applications. Tα1 can enhance T cells, dendritic cells and antibody responses, and also augment an anti-tumor immune response. In the current study, we developed a novel technique to produce Tα1 concatemer and investigated its capability in anti-tumor immunotherapy. We expressed the recombinant 2×Tα1 concatemer protein (Tα1② protein) in Escherichia coli. The purity of Tα1② was higher than 95% as assessed by HPLC analysis. In vitro, Tα1② could stimulate the proliferation of mouse splenic lymphocyte, and increase the apoptosis of tumor cell lines. In vivo, Tα1② significantly inhibited the tumor growth in B16 tumor-bearing mice. Compared with Tα1, the Tα1② is of more effective bioactivity than Tα1. The purified Tα1② is a promising substitute for synthetic Tα1 because of its potent anti-tumor effects. We concluded that the expression system for Tα1 concatemer was constructed successfully, which could serves as a highly efficient tool for the production of large quantities of the highly active protein.     

α-Tocopheryl phosphate: uptake, hydrolysis, and antioxidant action in cultured cells and mouse

α-Tocopheryl phosphate (α-TP), a water-soluble analogue of α-tocopherol, is found in humans, animals, and plants. α-TP is resistant to both acid and alkaline hydrolysis and may exert its own function in this form in vivo. In this study, the uptake, hydrolysis, and antioxidant action of α-TP were measured using α-TP with a deuterated methyl group, CD(3), at position 5 of the chroman ring (α-TP(CD3)). The hydrolysis of α-TP(CD3) was followed by measuring α-tocopherol containing the CD(3) group, α-T(CD3), in comparison to unlabeled α-tocopherol, α-T(CH3). α-TP(CD3) was incubated with cultured cells, and the intracellular α-T(CD3) formed was measured with HPLC-ECD and GC-MS. α-TP(CD3) was also administered to mice for 4 weeks by mixing in the diet, and α-T(CD3) was measured in plasma, liver, brain, heart, and testis to compare with endogenous unlabeled α-T(CH3). It was found that α-TP(CD3) was taken in and hydrolyzed readily to α-T(CD3) in cultured cells and in mice. The hydrolysis of α-TP(CD3) in cell culture medium was not observed. α-TP protected primary cortical neuronal cells from glutamate-induced cytotoxicity, and α-TP given to mice reduced the levels of lipid peroxidation products in plasma and liver. These results suggest that α-TP is readily hydrolyzed in vivo to α-T, which acts as an antioxidant, and that α-TP may be used as a water-soluble α-T precursor in intravenous fluids, in eye drops, or as a dietary supplement.     

The Bead blot: a method for selecting small molecule ligands for protein capture and purification

We present a new method for selecting peptide ligands that are useful for protein purification, protein targeting and exploring protein-ligand interactions, and which requires no prior protein purification or derivatization. In the Bead blot, a complex mixture containing the target protein, for example, plasma, is incubated with a combinatorial library of peptide ligands synthesized on beads. The proteins are fractionated and purified on their respective ligands and the beads with their bound proteins are immobilized in a gel. The proteins are eluted from the ligands by capillary action and captured on a membrane so that their position on the membrane corresponds to the position of the beads in the gel. The protein is detected on the membrane, generating spots on an autoradiography film, the spots on the film are aligned with the beads in the gel, and the beads that bound the protein are recovered. The ligand on the bead(s) can be sequenced and synthesized at large scale for protein purification. The Bead blot can be completed in several hours with overnight pause steps if desired. On average, 5 prospective ligands are selected per 50,000 beads screened using this method. Unlike other ligand identification methods, the target protein does not have to be purified or labeled, and the Bead blot allows ligands for multiple proteins to be selected in a single experiment.     

Novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association

This work combines two well-established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self-cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB-specific binding protein, a self-contained protein expression and purification system has been developed. In this system, the PHB-binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB-binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self-cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large-scale production of purified proteins and enzymes and high-throughput proteomics studies of peptide libraries.     

利用小肽控制的基于蛋白质内含子非层析标签制备重组蛋白(英文)

基于蛋白质内含子的蛋白质纯化自我断裂标签已经被广泛使用超过15年之久.但这一系统体内表达过程的提前断裂一直是限制这一技术广泛应用的瓶颈,特别是在需要高温表达和长表达周期的真核表达系统中.本研究介绍了一种利用小肽控制的基于蛋白质内含子和非层析标签ELP(elastin-like polypeptide)的自我断裂系统.在这一系统中,蛋白质内含子的体内外活性严格受到其结构互补小肽控制.在体内表达不含有互补小肽时,蛋白质内含子不具有活性;而在体外添加结构互补小肽,蛋白质内含子结构恢复并发生C端断裂反应释放目的蛋白.由于非层析标签ELP的引入,因此整个纯化过程可以简单地通过几步机械沉淀完成.此外,这一系统反应pH、小肽与前体蛋白之间的摩尔比及断裂速率也一并进行了系统的研究.     

Formation of chitin-based nanomaterials using a chitin-binding peptide selected by phage-display

Targeting polymers with peptides is an efficient strategy to functionalize biomaterials. Phage display technology is one of the most powerful techniques for selecting specific peptides for a wide variety of targets. A method to select a chitin-binding peptide from a 12-mer random peptide library was successfully performed against chitin immobilized in wells of microtiter plates. The synthetic chitin binding peptide (ChiBP) could bind to chitin beads and disrupt their structure. This selected peptide was successfully used to immobilize alkaline phosphatase on chitin. In addition, the peptide could induce colloidal chitin in water to form a chitin coat on the surface of plastic tubes. Scanning electron microscopy (SEM) revealed that the peptide could induce colloidal chitin and chitohexaose to form networks when the temperature was raised to 42°C.     

重组环状胸腺五肽结构类似物Cyclo-（Cys- Arg-Lys –Asp-Val-Tyr）的制备、鉴定和活性检测

为了实现蛋白内含肽(Intein)介导的重组环状胸腺五肽结构类似物［cyclo-（Cys -Arg-Lys –Asp-Val-Tyr）,cTP］的高效制备,设计并合成编码6个氨基酸的cTP基因,克隆到表达载体pTWIN1,重组表达质粒pTW-cTp转化E.coli ER2566构建工程菌,IPTG诱导由几丁质结合域纯化标签（chitin binding domain,CBD）、2个蛋白内含肽和目的多肽组成的“多元”融合蛋白(CBD-intein1-cTP-intein2-CBD)的高效表达.几丁质柱亲合层析纯化融合蛋白后,改变pH值和温度诱导intein1 C端切割,硫醇MESNA诱导intein2 N端切割,释放N端为Cys,C端为硫酯的重组cTP线性前体,通过非保护多肽硫酯环合法实现环肽生成.激光飞行质谱结果显示,纯化产物的分子量为764.4,与环肽的理论值相符.免疫活性检测结果显示,环肽cTP较线性多肽TP-5具有更显著的促进巨噬细胞吞噬能力的活性（P<0.01）和促进B细胞抗体生成的活性（P<0.01）.     

Cleavage and purification of intein fusion proteins using the Streptococcus gordonii spex system

A gram-positive bacterial expression vector using Streptococcus gordonii has been developed for expression and secretion, or surface anchoring of heterologous proteins. This system, termed Surface Protein Expression system or SPEX, has been used to express a variety of surface anchored and secreted proteins. In this study, the Mycobacterium xenopi (Mxe) GyrA intein and chitin binding domain from Bacillus circulans chitinase Al were used in conjunction with SPEX to express a fusion protein to facilitate secretion, cleavage, and purification. Streptococcus gordonii was transformed to express a secreted fusion protein consisting of a target protein with a C-terminal intein and chitin-binding domain. Two target proteins, the C-repeat region of the Streptococcus pyogenes M6 protein (M6) and the nuclease A (NucA) enzyme of Staphylococcus aureus, were expressed and tested for intein cleavage. The secreted fusion proteins were purified from culture medium by binding to chitin beads and subjected to reaction conditions to induce intein self-cleavage to release the target protein. The M6 and NucA fusion proteins were shown to bind chitin beads and elute under cleavage reaction conditions. In addition, NucA demonstrated enzyme activity both before and after intein cleavage.     

Characterization of a nucleus-encoded chitinase from the yeast Kluyveromyces lactis

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin alpha-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.     

Regulation of Mycobacterium-specific mononuclear cell responses by 25-hydroxyvitamin D3

The active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to be an important regulator of innate and adaptive immune function. In addition, synthesis of 1,25(OH)(2)D(3) from 25-hydroxyvitamin D(3) (25(OH)D(3)) by the enzyme 1α-hydroxylase in monocytes upon activation by TLR signaling has been found to regulate innate immune responses of monocytes in an intracrine fashion. In this study we wanted to determine what cells expressed 1α-hydroxylase in stimulated peripheral blood mononuclear cell (PBMC) cultures and if conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) in PBMC cultures regulated antigen-specific immune responses. Initially, we found that stimulation of PBMCs from animals vaccinated with Mycobacterium bovis (M. bovis) BCG with purified protein derivative of M. bovis (M. bovis PPD) induced 1α-hydroxylase gene expression and that treatment with a physiological concentration of 25(OH)D(3) down-regulated IFN-γ and IL-17F gene expression. Next, we stimulated PBMCs from M. bovis BCG-vaccinated and non-vaccinated cattle with M. bovis PPD and sorted them by FACS according to surface markers for monocytes/macrophages (CD14), B cells (IgM), and T cells (CD3). Sorting the PBMCs revealed that 1α-hydroxylase expression was induced in the monocytes and B cells, but not in the T cells. Furthermore, treatment of stimulated PBMCs with 25(OH)D(3) down-regulated antigen-specific IFN-γ and IL-17F responses in the T cells, even though 1α-hydroxylase expression was not induced in the T cells. Based on evidence of no T cell 1α-hydroxylase we hypothesize that activated monocytes and B cells synthesize 1,25(OH)(2)D(3) and that 1,25(OH)(2)D(3) down-regulates antigen-specific expression of IFN-γ and IL-17F in T cells in a paracrine fashion.     

一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析

目的：旨在分离并选择一株香蕉内生细菌作为内生基因工程生防菌,并克隆其几丁质酶基因的信号肽序列。方法：从香蕉植株杆下部分离并选择了一株拮抗香蕉枯萎病且具有分泌几丁质酶能力的内生细菌,对该菌株进行了形态观察、生理生化测定和16S rDNA序列分析,克隆了其几丁质酶基因的编码序列并预测了其信号肽,构建了含有信号肽和不含信号肽的几丁质酶的表达菌株BL-chi1和BL-chi2。结果：结合形态观察、生理生化特征和16S rDNA序列比对分析确定该菌株为Klebsiella属,将该菌株命名为KKWB 5;BL-chi1和BL-chi2经IPTG诱导后,均表达了与预期蛋白大小一致的蛋白,同时BL-chi1诱导后的培养基上清中出现一条约45kDa的条带,而BL-chi2和空载体的BL-pET22b诱导后的培养基上清中均无此条带;几丁质水解试验发现,BL-chi1诱导后的培养基上清中的蛋白经浓缩和纯化后都能在几丁质平板上形成透明水解圈。结论：该几丁质酶的信号肽能被BL21（DE3）所识别,将几丁质酶分泌到培养基中,并且分泌的几丁质酶具有水解几丁质的生物学活性。内生菌KKWB-5的分离及其几丁质酶分泌信号肽序列的克隆为进一步构建内生工程菌来防治香蕉枯萎病打下了基础。     

重组血管活性肠肽的制备及鉴定

为利用基因工程技术获得重组血管活性肠肽(vasoactive intestinal peptide,VIP),根据大肠杆菌的密码偏好性,设计并人工合成编码28个氨基酸的VIP基因。克隆到表达载体PTWIN,构建重组质粒PTWIN-VIP,转化宿主菌E. coli Strain ER2566,构建表达工程菌。实现由重组VIP,内含肽与纤维素结合域（cellulose binding domain, CBD）组成的融合蛋白表达。融合蛋白经几丁质亲和层析纯化,通过改变温度和缓冲液PH值切割融合蛋白,获得目的多肽。所得的多肽经质谱测定分子量结果与理论值相符。生物活性分析表明,重组VIP能显著降低急性炎症小鼠血清中抵抗素的水平,发挥抗炎作用。重组VIP的制备及其抗炎活性的鉴定为其深入开发奠定了基础。     

Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate

Adipogenesis inhibitory peptide was isolated and identified from black soybean (Rhynchosia volubilis Lour.) hydrolysate. An adipogenesis inhibitor was purified using consecutive methods including: ultrafiltration (MWCO; 3 and 10kDa), gel filtration chromatography (Superdex Peptide 10/300 GL column), and reverse-phase high-performance liquid chromatography (microBondapak C(18) column). Also, the adipogenesis inhibition effect of the purified peptide was measured by observation of droplet of 3T3-L1 adipocyte by Oil Red O staining in the highest active fraction in each step. The peptide was shown to inhibit the differentiation of the 3T3-L1 pre-adipocyte, which was confirmed by morphological study. The adipogenesis inhibitory peptide was purified 71.43-fold from black soybean hydrolysate throughout a five-step purification procedure. The adipogenesis inhibitor was identified to be a tripeptide, Ile-Gln-Asn, having an IC(50) value of 0.014 mg protein/ml. Furthermore, the synthetic tripeptide (Ile-Gln-Asn) exhibited the similar adipogenesis effects to the purified peptide. Thus, these results showed the potential anti-obesity effect of the purified peptide through control of adiposity.