Overexpression and characterization of the Rhodobacter sphaeroides PufX membrane protein in Escherichia coli

Heterologous expression of the PufX membrane protein from purple photosynthetic bacterium Rhodobacter sphaeroides was attempted by using Escherichia (E.) coli cells. The PufX was overexpressed as a recombinant protein with a histidine tag added to the carboxyl terminus, and can be extracted from the cell membrane by various detergents. Circular dichroism measurements showed that the expressed PufX protein had alpha-helix contents of 29% in organic solvents and 22-26% in 0.8-2.0% (w/v) n-octyl beta-D-glucopyranoside solutions, suggesting that the PufX contains a substantial alpha-helical region composed of 18-22 amino acids. The PufX expressed in E. coli was examined by reconstitution experiments with LH1 alpha- and beta-polypeptides and bacteriochlorophyll a. It was shown that the PufX inhibited not only the reconstitution of the LH1 complex, but also the formation of the B820 subunit type complex at high concentrations, indicating that the expressed PufX is biologically active. Large-scale expression of the functional PufX membrane protein provides sufficient quantity for further biophysical and structural analyses of its biological function, and adds another example for producing highly hydrophobic integral membrane proteins using the E. coli expression system.     

Native conformation at specific residues in recombinant inclusion body protein in whole cells determined with solid-state NMR spectroscopy

Inclusion bodies are insoluble aggregates that are formed by bacteria to store excess recombinant protein produced during expression. The structure of the protein in inclusion bodies is poorly understood but it has been hypothesized that the protein may form misfolded beta sheet aggregates. This paper presents an isotopic labeling and solid-state nuclear magnetic resonance approach to determine the secondary structure of individual residues within a recombinant influenza virus "FHA2" protein in inclusion bodies. The inclusion bodies were studied either in the context of the unlysed hydrated E. coli cells or in the hydrated pellet formed from centrifugation of the material insoluble in the cell lysate. The native structure of FHA2 is predominantly helical and native helical structure was also observed for several specific residues in the inclusion body FHA2. This approach will be applicable to structural analysis of many inclusion body proteins and should provide useful information for optimizing solubilization and purification protocols of these proteins.     

Semisynthesis and folding of the potassium channel KcsA

In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.     

Purification of a recombinantly produced transmembrane protein (gp41) of HIV I

The transmembrane protein gp41, a component of the viral envelope of HIV I, and its analogue gp36 of HIV II are important antigens for the sensitive and specific detection of anti-HIV antibodies. The immunodominant region of the protein gp41, which reacts with 100% of sera of infected persons, was produced by gene technological means in Escherichia coli. The protein accumulates in the form of insoluble inclusion bodies in the bacterial cell. Purification strategies for this aggregated material depend mainly on the isolation of these "inclusion bodies" and subsequent washing procedures. Growth conditions of the recombinant E. coli cells and the method of the cell disruption are important for the efficiency of purification and the recovery of the antigen. Owing to the insolubility of the expressed antigen, a significant concentration of recombinant gp41 was possible by extracting the soluble cell components. For this purpose, mild detergent solutions and low-molarity chaotropic buffer solutions were used. After final solubilization in 8 M urea buffer at pH 12.5, further chromatographic purification steps followed. The reduction of disulphide bridges with beta-mercaptoethanol or dithiothreitol was important. Gel filtration on a Sephacryl S-200 or Superose 12 column and/or ion-exchange chromatography on a DEAE-Sepharose Fast Flow or Mono Q HR (5/5) column finally resulted in the desired purity of the antigen.     

"Bioelectronic super-taster" device based on taste receptor-carbon nanotube hybrid structures

We have developed a method to monitor the activities of human taste receptor protein in lipid membrane using carbon nanotube transistors, enabling a "bioelectronic super-taster (BST)", a taste sensor with human-tongue-like selectivity. In this work, human bitter taste receptor protein expressed in E. coli was immobilized on a single-walled carbon nanotube field effect transistor (swCNT-FET) with the lipid membrane. Then, the protein binding activity was monitored using the underlying swCNT-FET, leading to the operation as a BST device. The fabricated BST device could detect bitter tastants at 100 fM concentrations and distinguish between bitter and non-bitter tastants with similar chemical structures just like a human tongue. Furthermore, this strategy was utilized to differentiate the responses of taster or non-taster types of the bitter taste receptor proteins.     

A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method.     

蛋白折叠液相色谱法复性和纯化大肠杆菌表达的重组人粒细胞集落刺激因子

用蛋白折叠液相色谱法(PFLC)对大肠杆菌表达的包涵体形式的重组人粒细胞集落刺激因子(rhG-CSF)进行了复性并同时纯化。用Cu2+-亚氨基二乙酸（IDA） Sepharose作为固定化金属离子亲和色谱的固定相。在低浓度脲存在下,以咪唑为洗脱剂,采用线性梯度洗脱rhG-CSF。该法仅通过一步PFLC分离,减少了复性过程中rhG-CSF的聚集,复性后的rhG-CSF的比活性为1.8×108 IU/mg,纯度为97%,质量回收率为32%。     

Purification of a crystallin domain of Yersinia crystallin from inclusion bodies and its comparison to native protein from the soluble fraction

It has been established that many heterologously produced proteins in E. coli accumulate as insoluble inclusion bodies. Methods for protein recovery from inclusion bodies involve solubilization using chemical denaturants such as urea and guanidine hydrochloride, followed by removal of denaturant from the solution to allow the protein to refold. In this work, we applied on-column refolding and purification to the second crystallin domain D2 of Yersinia crystallin isolated from inclusion bodies. We also purified the protein from the soluble fraction (without using any denaturant) to compare the biophysical properties and conformation, although the yield was poor. On-column refolding method allows rapid removal of denaturant and refolding at high protein concentration, which is a limitation in traditionally used methods of dialysis or dilution. We were also able to develop methods to remove the co-eluting nucleic acids during chromatography from the protein preparation. Using this protocol, we were able to rapidly refold and purify the crystallin domain using a two-step process with high yield. We used biophysical techniques to compare the conformation and calcium-binding properties of the protein isolated from the soluble fraction and inclusion bodies.     

重组鼠疫汉坦钩端多抗原位点同源合成基因的表达及活性鉴定

将人工合成的含鼠疫耶尔森氏菌的2个抗原位点、汉坦病毒的5个抗原位点和钩端螺旋体的5个抗原位点的串联基因片段克隆到原核表达载体pET-20b中,构建重组表达质粒pET-rYHL.转化E.coli BL21(DE3)后获得表达菌株.表达菌株经IPTG诱导后,可高效表达带组氨酸标签的以包涵体形式存在的融合蛋白.包涵体蛋白经尿...     

In situ structural characterization of a recombinant protein in native Escherichia coli membranes with solid-state magic-angle-spinning NMR

The feasibility of using solid-state magic-angle-spinning NMR spectroscopy for in situ structural characterization of the LR11 (sorLA) transmembrane domain (TM) in native Escherichia coli membranes is presented. LR11 interacts with the human amyloid precursor protein (APP), a central player in the pathology of Alzheimer's disease. The background signals from E. coli lipids and membrane proteins had only minor effects on the LR11 TM resonances. Approximately 50% of the LR11 TM residues were assigned by using (13)C PARIS data. These assignments allowed comparisons of the secondary structure of the LR11 TM in native membrane environments and commonly used membrane mimics (e.g., micelles). In situ spectroscopy bypasses several obstacles in the preparation of membrane proteins for structural analysis and offers the opportunity to investigate how membrane heterogeneity, bilayer asymmetry, chemical gradients, and macromolecular crowding affect the protein structure.     

Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts

The expression of classical swine fever virus (CSFV) structural protein E2 in different vectors, which has been shown to carry critical epitopes, has been established. Here, we reported a Chlamydomonas reinhardtii chloroplast expression vector, P64E2, containing classical swine fever virus structural protein E2 gene, which was constructed and transferred to C. reinhardtii by biolistic bombardment method. The transformants were identified by PCR, Southern blotting, Western blotting after selecting on resistant media. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.5-2% of the total soluble protein. The results of the study on the immunological activity indicated that the protein E2 expressed in C. reinhardtii chloroplasts could elicit animal bodies to produce antibodies against protein E2.     

小麦ACCase CT功能域基因在大肠杆菌中的表达及与除草剂的相互作用

以中国春小麦幼苗为材料,克隆构建了小麦质体乙酰辅酶A羧化酶(ACCase)的羧基转移酶(CT)重组质粒( RCP18-5),并实现了重组质粒在大肠杆菌中的可溶性高表达.对重组蛋白的性质研究表明,该蛋白具有较强的疏水性,稳定性不高.为改善这种状况,对CT功能域基因进行了截短和延长,同样于大肠杆菌中进行表达.结果表明,仅长...     

MMP-12 蛋白包涵体复性时折叠状态核磁共振图谱与荧光光谱研究

MMP-12 是癌症治疗药物靶标. 为了更好研制新药, 需要大量制备MMP-12, 但MMP-12 在大肠杆菌中以包涵体形式表达. 因此如何优化蛋白复性过程是大量获取MMP-12 蛋白的关键. 采用核磁共振、稳态荧光法、外源性ANS (8-anilinol-naphthalenesulfonic acid)荧光探针三种方法监控MMP-12 变性蛋白的再折叠过程, 以探究其复性折叠机制. 研究发现MMP-12 再折叠中点值与对应的尿素浓度几乎相等(C_m≈4, mid-point of transition). 不同尿素浓度中MMP-12 的二维¹H-¹⁵N HSQC (heteronuclear single quantum correlation)谱图显示, 尿素浓度从4 mol/L 降低到3 mol/L 是MMP-12 蛋白复性折叠的关键步骤. 据此我们将MMP-12 蛋白复性从常规的梯度透析复性方法改进成等容透析复性法(即确保尿素从4 mol/L 到3 mol/L 的浓度变化缓慢), 实现复性收率提高一倍.     

In Vitro Refolding of Triosephosphate Isomerase from <Emphasis Type="Italic">L. donovani</Emphasis>

The triosephosphate isomerase of Leishmania donovani (LdTIM) was expressed at high level in Escherichia coli. The TIM gene was cloned in expression vector pET-23(a) with C-terminal 6× His tag fused in frame, and expressed as a 27.6-kDa protein in E. coli as inclusion bodies. The recombinant LdTIM from E. coli lysate was solubilized in 6 M guanidine hydrochloride and purified by Ni-NTA chromatography. In the present study, the effect of bovine serum albumin on the reactivation of TIM was investigated. Furthermore, 8-anilino-1-naphthalene sulfonic acid was used to detect the structural changes induced by bovine serum albumin (BSA). Here, we conclude that BSA assists in the refolding and regain of LdTIM enzyme activity by providing framework for structure formation. This study indicates that numerous protein–protein contacts are constantly occurring inside the cell that leads to the formation of native protein.     

Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea, the recombinant enzyme was p...     

Strategies for examination of Alzheimer’s disease amyloid precursor protein isoforms

We describe a proteomics procedure using bioinformatics, immunoprecipitation, two-dimensional gel electrophoresis, Western blotting, in-gel digestion, LC–MS, MALDI–MS, and MS–MS for isolation and identification of amyloid precursor protein (APP) isoforms APP₆₉₅, APP₇₅₁, and APP₇₇₀. Retinoic acid-induced Ntera 2 cell line, derived from a human teratocarcinoma cells, was the in-vitro source of APP. Initial isolation of whole APP was performed by immunoprecipitation, using AB10, a monoclonal antibody raised to amino acids 1–17 of the β-amyloid peptide sequence, which is present in all three alpha secretase-cleaved isoforms of interest. The next stage was separation of whole APP into its isoform components by two-dimensional gel electrophoresis. Because of low APP concentrations, detection by the usual staining methods, for example Sypro Ruby, able to detect low picomole concentrations, did not enable visualisation of the isoforms. Western analysis, however, enabled primary detection of APP, because of the inherent sensitivity of antibodies raised to specific isoform regions. This initial visualization acted as a template for excision of isoforms from 2D gels, which were then subjected to peptide mass mapping. Initial theoretical digestion of each isoform revealed the presence of specific peptides, which were then used as “tags” for isoform detection.     

Enhanced In Vitro Refolding of Soluble Human Glucocorticoid-Induced TNF Receptor-Related Ligand

The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.     

Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease

We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.     

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration （ hALR）, Expression and Purification of hALR

Introduction Theliverisoneoftheorgansthathaspotentialre generativecapabilityinmammaliananimals[1].Studies oncaninemodelshaveindicatedthatthelivercanre generate,inonlytwoweeks,toitsoriginalsize,after70%hepatectomy[2].Therefore,theresearchoncellu larandmole…     

Gamma-Irradiation-Induced Degradation of the Water-Soluble Polysaccharide from Auricularia polytricha and Its Anti-Hypercholesterolemic Activity

The water-soluble polysaccharides (APPs) isolated from the edible mushroom Auricularia polytricha were irradiated by γ-ray at doses of 10, 100, and 1000 kGy. The effect of gamma irradiation on the degradation of the polysaccharide was investigated. After irradiation treatment, the viscosity and molecular weight of APPs decreased with the increase in the irradiation dose. The changes in the enthalpy of APPs after irradiation treatment were observed. Meanwhile, SEM showed that R-APPs were crushed into fragments and the surfaces became smooth and wrinkled after irradiation. In further spectrum analysis, it was found that the glycoside bonds of the polysaccharides were broken and accompanied by the formation of double bonds. This suggested that gamma irradiation could cause the depolymerization and oxidation of polysaccharides. In addition, irradiated APPs could reduce the body weight of hyperlipidemia mice. The levels of serum and liver TC, TG, and serum LDH-c significantly decreased in hyperlipidemia mice after treatment by irradiated APPs. It indicated that gamma irradiation significantly improved the anti-hypolipidemic activity of APPs. The relationship between the physicochemical properties and hypolipidemic activity of polysaccharides was interpreted, which provides a theoretical basis for the further development of APP products. Gamma irradiation is a viable technology for macromolecular modification for degradation.