Three-step chromatographic purification of Cpr6, a cyclophilin from Saccharomyces cerevisiae

Cyclophilins constitute a group of peptidyl-prolyl cis-trans isomerases (PPIs), known to be involved in protein folding. Because of their ability to bind the immunosuppresant drug Cyclosporin A (CsA), they are also called immunophilins. Immunophilins, which exhibit a relative molecular mass higher than 40 000, are further found in complex with Hsp90, a major cytosolic molecular chaperone. The present work describes a three-step chromatographic purification of recombinant Cpr6, a cyclophilin from Saccharomyces cerevisiae. The cDNA of Cpr6 was cloned into a pRSET A-plasmid with an N-terminal 6 x histidine-tag (his-tag) and transformed into the BL21[DE3]pLysS strain. After collection of the bacterial material and lysis of the cells the cell lysate was centrifuged and loaded onto a metal chelating column. After extensive washing the protein was eluted with a step gradient from 20 to 250 mM imidazol. The pooled protein was dialysed against ethylenedinitrilo tetraacetic acid (EDTA)-buffer, and loaded onto a strong anion-exchanger. Cpr6 containing fractions were then, in a last step, loaded onto a gel permeation chromatography column. The purity of the resulting protein was measured by silver stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and, additionally, as Cpr6 does not contain tryptophan residues by tryptophan residue titration. Based on a standard curve the content of contaminating tryptophan residues in the purified protein solution was determined. A typical yield of 1 mg pure protein per g of wet cells was achieved with the described procedure.     

Neuroprotective and antioxidant properties of FKBP-binding immunophilin ligands are independent on the FKBP12 pathway in human cells

We focused on immunophilin isoforms in order to clarify the neuroimmunophilins which were identified as targets for the immunophilin ligands to elicit a neuroprotective effect. Although the expressions of five FK506-binding protein (FKBP) mRNAs were detected in both SH-SY5Y (human neuroblastoma) and U251 (human glioma) cell lysates, the FKBP12 mRNA expression was detected in only the SH-SY5Y cells, and not the U251 cells. However, we found that the SH-SY5Y and the U251 cells were equipotent in the intensity of cellular protection of FK506 (an immunosuppressive immunophilin ligand) and GPI1046 (a non-immunosuppressive FK506 analog), indicating that the protective effect and glutathione activation of FK506 and GPI1046 had little need to bind FKBP12. Therefore, we conclude that the neuroprotective and antioxidant properties of immunophilin ligands are independent on the FKBP12 pathway.     

抗人FKBP52分子单克隆抗体的制备和鉴定

目的；研制抗人FKBP52单克隆抗体（mAb)，并分析其免疫生物学特性。方法：采用纯化的人FKBP52蛋白免疫BALB／c小鼠。用ELISA法鉴定mAb的特异性；用Western blot鉴定制备的9株mAb所识别的抗原相对分子质量（Mr)及结合人FKBP52分子的功能区。结果：共获得9株分泌抗人FKBP52 mAb的杂交瘤细胞。抗人FKBP52 mAb可识别相对Mr为52000在原核系统中表达的人FKBP52蛋白，也可识别Jurkat细胞浆中Mr为52000的天然FKBP52蛋白。两株mAb均可识别人FKBP52分子中的第2功能区。其余7株mAb可识别人FKBP52分子中的第3功能区。结论：成功地制备了抗人FKBP52分子mAb。     

Three-step purification of bacterially expressed human single-chain Fv antibodies for clinical applications

We have obtained a cell line which secretes a human monoclonal IgM (B7) reacting with the myosin heavy chain of human heart. We have constructed single-chain fragments (scFv) of B7. The scFv may be useful for the imaging of myocardial necrosis after myocarditis, cardiac drug toxicosis or graft rejection. The aim of our work was to purify the scFv for immunoscintigraphy. We describe several purification steps including immobilized metal affinity chromatography (IMAC), anti-c-myc monoclonal antibody affinity chromatography, size-exclusion chromatography with Superdex 75 HR 10/30 and ion-exchange chromatography (mini Q TM 30Q).     

Refolding and purification of a urokinase plasminogen activator fragment by chromatography

A fragment of recombinant urokinase plasminogen activator (u-PA), was expressed in E. coli in the form of inclusion bodies. Purification and renaturation was achieved in a three-stage process. Capture of the inclusion bodies was achieved by coupling wash steps in Triton X-100 and urea with centrifugation. Solubilised inclusion bodies were then renatured by buffer exchange performed by size-exclusion chromatography (SEPROS). Use of size-exclusion media with higher fractionation ranges resulted in an increase in the recovery of u-PA activity, to a maximum fractionation range of Mr 10000-1500000 after which recovery is reduced, due to a low resolution between the refolded u-PA and denaturant. Fractions of refolded u-PA were concentrated using cation ion-exchange chromatography, which selectively binds correctly folded u-PA. The result is concentrated, active, homogeneous u-PA.     

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by approximately 25-fold in WT MEFs, but only by approximately 4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency approximately 23-fold in WT MEFs, but only approximately 4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, approximately 59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only approximately 28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.     

炭疽中性蛋白酶部分片段的重组表达、纯化及免疫效果研究

构建表达炭疽杆菌中性蛋白酶基因部分片段N蛋白(位于248~532氨基酸),并对其免疫效果进行研究;设计引物采用PCR法自炭疽杆菌扩增中性蛋白酶部分片段N,T-A克隆后构建原核表达质粒pET28a(+)-N,通过Western blot检测重组蛋白的免疫原性。以Al(OH)3为佐剂皮下注射免疫BALB/c小鼠,用ELISA方法测血清总IgG水平,MTT比色法测定脾T淋巴细胞增殖,采用流式分选法对脾T淋巴细胞亚群分类;Western blot显示重组蛋白具有免疫原性,rN免疫BALB/c小鼠后,ELISA检测到免疫鼠体内有特异性抗体产生;T淋巴细胞亚群试验表明,重组N蛋白以诱发Th2型免疫反应为主;MTT试验表明,rN蛋白对小鼠脾细胞在体外有促进生长和增殖能力,说明该重组蛋白具有生物学活性。纯化的rN可诱导高水平的抗体反应,为进一步的功能研究奠定了基础。     

Evidence of association between FKBP1B and thyroid autoimmune disorders in a large Tunisian family

FKBP1B belongs to immunophilins superfamily and functions as a cytosolic receptor protein of FK506. The role of FKBP1B in the immunosuppressive pathway of FK506 is well established. Previously, we reported a strong evidence of linkage between D2S171 microsatellite marker (located in vicinity of FKBP1B gene) and susceptibility to autoimmune thyroid diseases (AITDs). In this study, we report linkage disequilibrium between the dimorphism (C/T) in the 3' untranslated region (3' UTR) of FKBP1B gene and susceptibility to AITDs. DNAs were extracted from a large Tunisian family affected with Graves' disease (GD) and Hashimoto's thyroiditis (HT) and analysed by PCR-RFLP using DraIII restriction enzyme. Our results showed an excess of transmission of the allele C from heterozygous parents to affected offspring (transmission disequilibrium test (TDT) = 4.76; p = 0.012). This suggests a linkage disequilibrium of 3' UTR (C/T) SNP with AITDs. Moreover, The FBAT analysis gives a significant association with the C allele under the recessive model (chi2 = 5.50; p = 0.018). These results support the involvement of FKBP1B gene in the genetic susceptibility to the AITDs development in the studied family.     

Autumnalamide targeted proteins of the immunophilin family

Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ionomycin Ca²⁺ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca²⁺ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca²⁺ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca²⁺ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The K_D obtained for Cyp D- autumnalamide was 1.51 ± 1.399. Moreover, the K_D for Cyp A- autumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08 ± 1.23 and 6.85 ± 1.1 μM respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca²⁺ channels.     

Cloning and characterisation of a large metagenomic DNA fragment containing glycosyl-hydrolase genes

The problem of searching for and characterizing enzymes produced by uncultured microorganisms is presently settled by creating metagenomic libraries. A 6000-clone library with the average size of inserts of about 15 kb has been constructed based on total DNA isolated from cow rumen microorganisms. As a result of library screening on plates with different substrates, a clone was selected that efficiently hydrolysed lichenan and carboxymethylcellulose. The clone contained the recombinant plasmid pBlue-13 bearing a 12071 bp-long metagenomic fragment carrying ten open reading frames, two of which were identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any sequences known microorganism genomes was revealed. The amino acid sequence deduced from frame 4 was denoted of Xyl3A, and bears resemblance with β-xylosidases of glycosyl hydrolase family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydorlase family 5. The amino acid sequences deducted from on seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family and the bacteria inhabiting mammalian intestines.     

An amino-terminal fragment of SV40 T antigen transforms REF52 cells.

An SV40 mutant, T147D, encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We show here that a retrovirus which expresses this mutant T antigen transforms rat REF52 cells as efficiently as a retrovirus that expresses both the wild-type large and small T antigens. This cell line had previously been refractory to transformation by mutants that make short, amino-terminal fragments of T antigen.     

Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides

Glycoprotein 96 (gp96), a member of heat shock protein (HSP) family, plays important roles in both innate immunity by itself and in T cell adaptive immunity in complex with antigenic-specific peptides. Using ammonium sulfate precipitation, ConA-Sepharose affinity chromatography and anion exchange chromatography, we have successfully purified gp96 from human liver tumor tissues. Subsequently, the gp96-associated peptides were successfully isolated from the gp96 preparation in quantities sufficient for micro-sequencing and identification of the peptides [Lancet 357 (2001) 528]. This three-step purification method might be used as a universal technique for the easy and reproducible isolation of antigenic heat shock protein gp96 from liver tissue or even other tissues of different sources. This will inevitably trigger the search for antigenic-specific peptides bound to gp96. Here, we report the method in detail.     

Functional folding of a cytoplasmic single-chain variable fragment and its use as elutable protein purification tag

The variable fragment (Fv) of the monoclonal B1-8 antibody recognizes 3-nitro-4-hydroxy-phenylacetate (NP) and 5-iodo-NP (NIP) allowing for the affinity purification of the respective B cell antigen receptor with NP-sepharose and its specific elution with NIP-capronic acid (NIPcap). We generated an intracellular single-chain B1-8 Fv (iscFv), fused it to the N-terminus of the regulatory subunit (p85alpha) of phosphatidylinositol-3-kinase (PI3K) (isc-p85alpha), and examined the potential of this iscFv to serve as an intracellular elutable protein purification tag. The isc-p85alpha fusion protein could be specifically affinity-purified from the lysates of transfected Drosophila S2 cells with NP-sepharose and eluted with NIPcap, indicating the functional folding of the iscFv in the reducing environment of the cytosol. Furthermore, co-purification of the catalytic subunit of PI3K (p110) was achieved from lysates of co-transfected S2 cells as well as RBL-2H3 mast cells stably expressing isc-p85alpha. This indicates that the iscFv part of isc-p85alpha does not negatively influence p85alpha folding and interaction with p110. Moreover, successful incorporation of the p85alpha-moiety of isc-p85alpha into endogenous protein complexes in mast cells suggests the use of isc-containing fusion proteins for the native purification, elution, and analysis of intracellular signaling complexes.     

SARS冠状病毒膜蛋白膜内区的表达、纯化及抗原性初步研究

目的对SAILS冠状病毒膜蛋白膜内区基因片段进行克隆表达和表达产物的纯化复性,探讨该表达蛋白的抗原特性。方法克隆SAILS冠状病毒GD322株膜蛋白膜内区,在原核表达系统中表达His-融合蛋白,采用Western blot和酶联免疫吸附试验(ELISA)分析His-融合蛋白抗原特性。结果7份SARS临床诊断患者血清中5份血清能与His-融合蛋白反应,2份不与之反应。兔抗OCA3、229E血清及20份健康人血清皆不与His-融合蛋白反应。His-融合蛋白免疫动物可产生多克隆抗体。结论本研究获得的His-融合蛋白可与SARS临床诊断患者血清特异性结合,可免疫动物获得特异性多抗;但不与健康人血清及兔抗OCA3、229E血清发生特异性结合。     

Conditional immortalization of rodent fibroblasts using a temperature sensitive mutant fragment of polyoma virus large T antigen

We describe a method for prolonging the life span of primary cells using an N-terminal fragment of a temperature sensitive mutant of the polyoma virus oncogene, its large T antigen (LT). This allows long term characterization studies to be carried out on cells which would otherwise have senesced after a few passages in culture. Further, repeated preparation of cells from tissue sources can be avoided. Moreover, the cells can also be reverted to a mortal state, if desired, by incubating at the non-permissive temperature for the LT function.     

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments.     

TLR4胞外段在大肠杆菌中的可溶性表达、纯化及鉴定

目的：构建谷胱甘肽转硫酶-TLR4融合蛋白表达载体,并研究其编码的蛋白质在大肠杆菌中的表达。方法：利用PCR从含TLR4全长cDNA序列的质粒中扩增其胞外段,约1800bp,然后将其克隆入pMD18-T载体中。测序确证后重组入谷胱甘肽转硫酶融合表达载体pGEX-4T-1中,并用IPTG诱导其在大肠杆菌中表达。结果：PCR扩增的TLR4胞外区基因序列与GenBank数据库中的序列一致。重组质粒经酶切鉴定及测序证实,TLR4基因已正确插入到pGEX-4T-1中。重组表达质粒转化感受态大肠杆菌HBl01后用IPTG诱导,获得Mr92 000的GST-TLR4融合蛋白;37℃,O.2mmol／L IPTG诱导4h,SDS-PAGE电泳后,经薄层凝胶扫描分析该融合蛋白占菌体蛋白总量34．25％,部分以包涵体存在,可溶性蛋白约占27．84％;亲和层析纯化后纯度大于90％。Western-blot证实GST-TLR4融合蛋白能与TLR4单抗特异性结合。结论：成功地构建融合蛋白表达载体pGEX-TLR4,并在大肠杆菌中获得有效表达,获得了纯度大于90％的可溶性GSI-TLR4融合蛋白,为进一步研究其功能及制备TLR4单克隆抗体奠定了基础。