TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.     

重组人白细胞介素-6的纯化

重组人白细胞介素－６（ｒｈＩＬ－６）在工程菌ｐＢＶ２２０／ｒｈＩＬ－６／ＤＨ５ａ中以包涵体形式高效表达。ｒｈＩＬ－６经过工程菌体破碎、包涵体分离及抽提、复性、色谱分离后得到高度纯化。纯化产物纯度９５％,具有良好的生物学活性     

Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli

To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.     

Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization

Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization. Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E. coli promoter. The transgenic E. coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E. coli, suggesting the synthesis of an active protein. The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species. The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E. coli through an unknown mechanism. The study elucidates a hitherto unknown possible function of PQQ in bacteria.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Proteolytic activity of HtpX, a membrane-bound and stress-controlled protease from Escherichia coli

Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.     

Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: comparison with the native protein from bovine brain

Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities of calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ser262 determines the chloride-dependent colour tuning of a new halorhodopsin from Haloquadratum walsbyi

Light is an important environmental signal for all organisms on earth because it is essential for physiological signalling and the regulation of most biological systems. Halophiles found in salt-saturated ponds encode various archaeal rhodopsins and thereby harvest various wavelengths of light either for ion transportation or as sensory mediators. HR (halorhodopsin), one of the microbial rhodopsins, senses yellow light and transports chloride or other halides into the cytoplasm to maintain the osmotic balance during cell growth, and it exists almost ubiquitously in all known halobacteria. To date, only two HRs, isolated from HsHR (Halobacterium salinarum HR) and NpHR (Natronomonas pharaonis HR), have been characterized. In the present study, two new HRs, HmHR (Haloarcula marismortui HR) and HwHR (Haloquadratum walsbyi HR), were functionally overexpressed in Escherichia coli, and the maximum absorbance (λmax) of the purified proteins, the light-driven chloride uptake and the chloride-binding affinity were measured. The results showed them to have similar properties to two HRs reported previously. However, the λmax of HwHR is extremely consistent in a wide range of salt/chloride concentrations, which had not been observed previously. A structural-based sequence alignment identified a single serine residue at 262?in HwHR, which is typically a conserved alanine in all other known HRs. A Ser262 to alanine replacement in HwHR eliminated the chloride-independent colour tuning, whereas an Ala246 to serine mutagenesis in HsHR transformed it to have chloride-independent colour tuning similar to that of HwHR. Thus Ser262 is a key residue for the mechanism of chloride-dependent colour tuning in HwHR.     

抗人CD19单链抗体基因的构建、表达及功能测定

采用RT-PCR方法从分泌抗人类白细胞表面分化抗原CD19单克隆抗体的杂交瘤细胞中克隆出V_H和V_L可变区基因,再通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人CD19单链抗体（抗CD19-ScFv）基因。将其克隆至表达载体PET28a并在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明,抗CD19-ScFv在BL21（DE3）菌中获得表达,重组蛋白的相对分子量为27kD,表达产物以不溶性包涵体形式存在,经过溶解包涵体,镍柱亲和层析纯化和体外复性过程,获得了高纯度的单链抗体片段。流式细胞分析结果证实抗CD19ScFv可与人类白细胞表面的分化抗原CD19结合,保留了鼠源性单抗与CD19结合活性。抗人CD19-ScFv的构建与表达,为下一步针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础。     

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.     

大肠杆菌表达重组人白细胞介素-9的纯化及部分性质

表达重组人白细胞介素－９（ｒｅｃｏｍｂｉｎａｎｔｈｕｍａｎｉｎｔｅｒｌｅｕｋｉｎ－９ｒｈＩＬ－９）的大肠杆菌经破碎、包涵体洗涤、裂解提取、凝胶过滤和离子交换色谱分离,得到了电泳纯的ｒｈＩＬ－９,回收率６６％,分子量与理论值相符,有刺激小鼠骨髓巨核系集落生成的活性．为ｒｈＩＬ－９的大规模制备及更深入的研究奠定了基础     

Engineering of a proteolytically stable human beta 2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells

The hydrophobic human beta 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli. Photoaffinity labeling with the adrenergic ligand [125I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli. The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other beta 2 adrenergic receptor constructs in E. coli. After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli. The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.     

异戊二烯合成酶(IspS)在大肠杆菌中的表达及其产异戊二烯的研究

将来源于银白杨的异戊二烯合成酶基因按照大肠杆菌密码子偏爱性进行优化,克隆到表达载体pACYCDu-et-1上,在大肠杆菌BL21(DE3)中异源表达,采用镍柱亲和层析纯化重组蛋白并测定其异戊二烯合成酶活性,通过摇瓶发酵实验对重组菌产异戊二烯进行进一步研究。结果显示:银白杨异戊二烯合成酶在大肠杆菌中能够高效表达,经过镍柱纯化后,电泳检测到特异性表达条带;该重组异戊二烯合成酶能够催化异戊二烯的合成,重组菌的异戊二烯产量可达到60μg/L。     

Phospholipid requirement and pH optimum for the in vitro enzymatic activity of the E. coli P-type ATPase ZntA

Detergent solubilization and purification of the E. coli heavy metal P-type ATPase ZntA yields an enzyme with reduced hydrolytic activity in vitro. Here, it is shown that the in vitro hydrolytic activity of detergent solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. The protein-lipid interaction of ZntA was characterized by enzyme-coupled ATPase assays and fluorescence spectroscopy. Among the most abundant naturally occurring phospholipids, only phosphatidyl-glycerol lipids (PG) enhance the in vitro enzymatic ATPase activity of ZntA. Re-lipidation of detergent purified ZntA with 1,2-dioleoylphosphatidyl-glycerol (DOPG) increases the ATPase activity four-fold compared to the purified state. All other E. coli phospholipids fail to activate the ATPase. Among the phosphatidyl-glycerol family, highest activity was observed for 1,2-dioleoyl-PG followed by 1,2-dimyristoyl-PG, 1,2-dipalmitoyl-PG and 1,2-distearoyl-PG. Increasing intrinsic Trp fluorescence quantum yield upon relipidation of ZntA was used to determine a pH maximum for lipid binding at pH 6.7. The pH dependence of the lipid binding was confirmed by pH-dependent ATPase assays showing maximum activity at pH 6.7. The biophysical characterization of detergent solubilized membrane proteins crucially relies on the conformational stability and functional integrity of the protein under investigation. The present study describes how the E. coli ZntA P-type ATPase can be stabilized and functionally activated in a detergent solubilized system.     

Conserved high affinity ligand binding and membrane association in the native and refolded extracellular domain of the human glycine receptor alpha1-subunit

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding alpha- and gephyrin anchoring beta-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (alpha1-(1-219)) and the large intracellular TM3-4 loop (alpha1-(309-392)) of the human GlyR alpha1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested approximately 25% alpha-helix and approximately 48% beta-sheet for the extracellular domain, while no alpha-helices were detectable for the TM3-4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of approximately 5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of approximately 49 kDa indicated oligomerization in a defined native structure. As shown by [(3)H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.     

Glucose dehydrogenase of a rhizobacterial strain of <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae

Glucose dehydrogenase (GDH) of Gram-negative bacteria is a membrane bound enzyme catalyzing the oxidation of glucose to gluconic acid and is involved in the solubilization of insoluble mineral phosphate complexes. A 2.4 kb glucose dehydrogenase gene (gcd) of Enterobacter asburiae sharing extensive homology to the gcd of other enterobacteriaceae members was cloned in a PCR-based directional genome walking approach and the expression confirmed in Escherichia coli YU423 on both MacConkey glucose agar and hydroxyapatite (HAP) containing media. Mineral phosphate solubilization by the cloned E. asburiae gcd was confirmed by the release of significant amount of phosphate in HAP containing liquid medium. gcd was over expressed in E. coli AT15 (gcd::cm) and the purified recombinant protein had a high affinity to glucose, and oxidized galactose and maltose with lower affinities. The enzyme was highly sensitive to heat and EDTA, and belonged to Type I, similar to GDH of E. coli.     

Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport

The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre-extraction of the membranes with 5-sulfosalicylate, solubilization in dodecyl-O-beta-D-maltoside, Ecteola-column chromatography, and removal of residual impurities by anti-impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles. Purification was monitored by tracer N-[3H]ethylmaleimide-labeled carrier and by binding of the substrate p-nitrophenyl-alpha-D-galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria. Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain. Large unilamellar proteoliposomes (1-5-micron diameter) were prepared from initially small reconstituted vesicles by freeze-thaw cycles and low-speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (delta mu H+) or one of its components (delta psi or delta pH). Comparison of the steady-state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport. The half-saturation constants for active uptake of lactose (KT = 200 microM) or beta-D-galactosyl-1-thio-beta-D-galactoside (KT = 50-80 microM) by the purified carrier were found to be similar to be similar to those measured in cells or cytoplasmic membrane vesicles. The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat = 3-4 s-1 for lactose) but considerably smaller than in cells (kcat = 40-60 s-1). Possible reasons for this discrepancy are discussed.     

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.     

人源抗TNF—α抗体Fab基因的单链化及其表达和纯化

构建人源抗TNF－α单链抗体基因,并尝试其在E.coli中的表达和纯化,采用人工接头,按VH－linkerVL的结构将人源抗TNF－a的VH,VL基因拼接成单链抗体（ScFv)基因,并将构建好的ScFv基因插入表达载体pQE30,转染E.coli M15,以IPTG诱导表达,Ni-NTA树脂纯化表达产物,构建的ScFv基因长723bp, 列分析表明,该序列拼接正确,SDS－PAGE显示,用重组的pQE30转化的M15菌经诱导后,有相对分子质量（M）约为52000的外源蛋白表达,Ni-NTA树脂纯化的表达产物纯度大于90％,因此,成功地构建了人源抗TNFa的ScFv基因,并在E.coli DH5a中表达和纯化的该基因的产物。     

Expression, Purification, and Encephalitogenicity of Recombinant Human Myelin Oligodendrocyte Glycoprotein

Abstract: Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N-terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST-MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS-like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.