Purification of an L-asparaginase-atrial natriuretic peptide fusion protein expressed in Escherichia coli

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.     

Conversion of beta-human atrial natriuretic polypeptide into alpha-human atrial natriuretic polypeptide in human plasma in vitro

Using synthetic beta-human atrial natriuretic polypeptide (beta-hANP), an antiparallel dimer of alpha-hANP, and radioimmunoassay (RIA) for alpha-ANP which also detects beta-hANP, we investigated the disappearance profile and the change in the molecular form of exogenously added beta-hANP in human plasma in vitro, compared with those of alpha-hANP. The ANP-like immunoreactivity (ANP-LI) level in beta-hANP-added human plasma exhibited slower disappearance than that in alpha-hANP-added plasma during the incubation at 37 degrees C. High performance-gel permeation chromatography and reverse phase-high performance liquid chromatography coupled with RIA revealed that beta-hANP (6K) was converted into a smaller peptide with an approximate molecular weight of 3K corresponding to alpha-hANP during the incubation. Amino acid analysis and amino-terminal sequencing confirmed that the converted peptide from beta-hANP in human plasma is authentic alpha-hANP. The demonstrated conversion of beta-hANP into alpha-hANP in human plasma could be relevant to the in vivo natriuretic and diuretic actions with slower onset and longer duration of this unique peptide.     

人胰岛素原类似物(BKRA)基因的合成与表达

为了利用基因工程生产胰岛素,按照已知的人胰岛素Ａ、Ｂ链氨基酸序列和大肠杆菌偏爱的氨基酸密码子设计并合成了人胰岛素原类似物（ＢＫＲＡ）基因,其中以赖（Ｋ）－精（Ｒ）二肽编码区取代人胰岛素原Ｃ肽编码区．为了避免其编码蛋白在大肠杆菌中表达时被降解,通过人工接头将２个ＢＫＲＡ基因串联起来,接头部分氨基酸序列为Ａｒｇ－Ａｒｇ－Ａｓｎ－Ｓｅｒ．将串联的ＢＫＲＡ基因克隆到表达载体ｐＥＴ－２８ａ（＋）,实现了在大肠杆菌中的融合表达,表达产物以包含体形式存在,约占细菌总蛋白２４％．表达产物氨基末端具有六组氨酸肽段,以ＨｉＴｒａｐ凝胶进行亲和层析,一步纯化可达纯度９５％以上．放射免疫测定表明,纯化的融合蛋白具有胰岛素抗原活性．表明已构建成人胰岛素原类似物的高效表达菌株     

High-yield production of recombinant endothelin-1.

A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography. Trypsin digestion of the purified pETB-42P gave big ET-1(1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1(1-37) gave ET-1(1-21) in a yield of 74% (overall yield: 52%). Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60%. One milligram of ET-1 or 2.0 mg of big ET-1(1-37) was obtained from 1.8 liters of culture broth. Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1(1-37) had almost the same in vitro and in vivo biological activities as big ET-1(1-38).     

Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.     

A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine

A highly sensitive radioimmunoassay has been established for measurement of human plasma and urine concentrations of atrial natriuretic peptide (ANP) and requires no extraction or concentration process such as Sep-Pak C-18 cartridge treatment. An antiserum was prepared from rabbits immunized with alpha-human ANP (alpha-hANP) coupled with bovine-thyroglobulin. The sensitivity of this method was 0.3 pg/tube of synthetic alpha-hANP utilized as authentic standard. Recovery of alpha-hANP spiked to plasma and urine was 97.7 +/- 15.4% and 97.1 +/- 9.5% (mean +/- SD), respectively. Plasma and urinary ANP concentrations versus assay data showed satisfactory linearity. In 124 healthy subjects, the plasma ANP-concentration was 31.7 +/- 12.0 pg/ml. Two different molecular forms of ANP in plasma and a single form in urine were found by gel permeation chromatography.     

Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes

Hepatic lipase, a glycoprotein synthesized and secreted by the hepatocyte, binds to sinusoidal endothelium where it is involved in metabolism of lipoprotein phospholipid and triglyceride. To better understand the regulation of hepatic lipase, we investigated the synthesis, post-translational processing, and secretion of the enzyme by isolated rat hepatocytes. Metabolically labeled [35S]methionine hepatic lipase protein, produced by the collagenase-dispersed hepatocytes, was immunoisolated from detergent-solubilized cells and incubation medium at designated times, using a polyclonal rabbit anti-rat hepatic lipase antibody raised against hepatic lipase purified to homogeneity from rat liver post-heparin perfusates. Following polyacrylamide gel electrophoresis and fluorography, radiolabeled hepatic lipase was quantitated by densitometry. Newly synthesized hepatic lipase was rapidly secreted and accumulated in the medium as a 59,000-dalton protein in a manner consistent with a constitutive process. An intracellular 53,000-dalton precursor of the mature 59,000-dalton hepatic lipase was identified by immunoprecipitation. The 53,000-dalton form could also be generated by endoglycosidase digestion of the secreted 59,000-dalton protein. In pulse-chase experiments, the 53,000-dalton protein was converted into the 59,000-dalton form. A 47,000-dalton form of hepatic lipase was immunoisolated from cell lysates only after tunicamycin treatment and could be generated from the secreted 59,000-dalton enzyme by prolonged endoglycosidase digestion. These data show that hepatic lipase is synthesized and rapidly secreted by isolated rat hepatocytes. Further, an intracellular 47,000-dalton precursor peptide can be identified after tunicamycin treatment, which may represent the hepatic lipase polypeptide, presumably after removal of its signal sequence; a 53,000-dalton partially glycosylated peptide exists as a major precursor form in the cell; and the mature 59,000-dalton hepatic lipase is present in the hepatocyte, but it is rapidly secreted.     

Structural organization of the lens fiber cell plasma membrane protein MP18

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.     

水通道蛋白1-C末端肽段/GST融合蛋白的原核表达(简报)

水通道蛋白(Aquaporin,AQP)是一类选择性高效转运水分子的细胞膜通道蛋白,广泛存在于原核和真核生物细胞的细胞膜上,主要介导自由水分子的被动跨膜转运,对保持细胞内外液环境的稳态平衡起着重要的作用.     

大肠杆菌中高效表达rhBMP-4的探讨及初步活性测定

目的 在大肠杆菌中表达具有生物活性的rhBMP-4。方法 在不改变氨基酸序列的前提下,以全基因合成的方式对人BMP-4成熟肽基因全长进行定点突变,将之重组入pET-3c表达载体并转化至大肠杆菌BL21(DE)plysS。IPTG诱导和包涵体复性后,利用C2C12细胞横向成骨细胞分化实验以及小鼠肌袋异位骨形成实验检测其活性。 结果 获得0.348 kb的BMP-4 DNA序列,表达的目的蛋白主要以包涵体的形式存在。经纯化及复性后,体内与体外的活性检测表明rhBMP-4有良好的诱骨生成活性。结论 该方案能够实现rhBMP-4在大肠杆菌中的高效表达。     

Purification and characterization of decorin from the culture media of MRC-5 cells

Myofibroblasts play an important role in fibrogenesis. Myofibroblasts secrete several components of the extracellular matrix, including decorin. To clarify the properties of decorin synthesized by myofibroblasts, we have purified and characterized decorin secreted into culture medium by the myofibroblast cell line MRC-5. Decorin was purified by successive chromatography steps using Hitrap Q and Superdex 200. Purified decorin showed a broad band on SDS-polyacrylamide gel electrophoresis, which was resolved into two smaller molecular weight bands after digestion with chondroitinase ABC. Further digestion with N-glycanase resolved these two bands into a single band, indicating that the N-glycation pattern of decorin is heterogeneous. The N-terminal amino acid sequence analysis of the purified protein and its reactivity towards an antibody raised against a C-terminal peptide of decorin indicate that MRC-5 cells secrete full-length decorin into the culture medium. To characterize the glycosaminoglycan chains attached to decorin, glycosaminoglycans from the purified protein were treated with chondroitinase ACI, chondroitinase ACII, chondroitinase ABC and chondroitinase B. The resulting disaccharides were analyzed by chromatography, which indicated that decorin secreted by MRC-5 cells is a dermatan sulfate proteoglycan. In conclusion, the decorin secreted by MRC-5 cells has similar characteristics to the decorin expressed in several tissues. Thus, culturing MRC-5 cells may be highly useful for studying the role of decorin and myofibroblasts in fibrosis.     

重组人胱抑素C的原核表达及鉴定

目的构建重组人胱抑素C（cystatinC,CysC）的原核高效表达质粒,诱导表达并纯化获得CysC重组蛋白。方法根据大肠埃希菌编码蛋白的特性设计CysC编码基因序列,人工合成目的基因克隆至pET-22b（＋）表达载体中,测序及酶切鉴定正确后诱导其在大肠埃希菌BL21中表达,所获得的包涵体蛋白经亲和层析纯化后采用SDS—PAGE及Western印迹鉴定。结果酶切结果证实构建的表达质粒结构正确;测序结果显示克隆的基因序列所编码的蛋白与GenBank中的CysC氨基酸序列相符;SDS-PAGE及Western印迹结果证实获得的重组CysC融合蛋白分子量约为16kD,经NP亲和层析纯化获得纯度大于90％的目的蛋白。结论建立了重组人CysC的原核高效表达系统并获得了CysC重组蛋白。     

Vacuolar targeting and posttranslational processing of the precursor to the sweet potato tuberous root storage protein in heterologous plant cells

Sporamin, the tuberous root storage protein of the sweet potato, which is localized in vacuoles, is synthesized as a prepro-precursor with an N-terminal sequence of amino acids that includes a signal peptide and an additional pro-segment of 16 amino acids. A full-length cDNA for sporamin was placed downstream of the 35 S promoter of cauliflower mosaic virus and introduced into tobacco and sunflower genomes by Ti plasmid-mediated transformation. A polypeptide of nearly the same size as mature sporamin from the sweet potato was detected in transformed calli of tobacco and sunflower, as well as in the leaves, stems, and roots of regenerated, transgenic tobacco plants. Amino acid sequence analysis of the nearly mature-sized form of sporamin from the transformed tobacco cells revealed that it is actually longer by three amino acids at its N terminus than authentic sporamin purified from the sweet potato. By pulse labeling of suspension-cultured tobacco cells with [35S]methionine, the pro-form of the precursor to sporamin, but not the prepro-precursor, was detected. The 35S-labeled proform was chased to the nearly mature-sized form via an intermediate form which is slightly larger than the nearly mature-sized form. Analysis by Edman degradation of the intermediate form that was labeled in vivo with [3H]histidine suggested that it is longer by two amino acids at its N terminus than the nearly mature-sized form of sporamin. These results suggest that at least two steps of posttranslational processing of the pro-form occurs sequentially in tobacco cells. The posttranslational processing of the pro-form of the precursor to sporamin was inhibited by monensin, suggesting that this step takes place in the acidic compartment, probably in the vacuole. All of the sporamin polypeptides synthesized in transformed tobacco cells were retained inside the cell and sporamin was localized in the vacuole, as judged from results of subcellular fractionation. These results indicate that sporamin is appropriately targeted to the vacuole in tobacco cells.     

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi: high level expression and purification of a soluble recombinant form of OspA

The ospA gene of Borrelia burgdorferi encodes an outer membrane protein which is a major antigen of the Lyme disease agent. Two sequence-specific sets of oligonucleotide primers were used to specify the amplification of the ospA coding sequence by the polymerase chain reaction. One set allowed the entire ospA sequence to be amplified, while the other primed amplification of a truncated form of ospA lacking the first 17 codons specified by the wild-type ospA structural gene, residues believed to constitute a signal sequence which normally would direct localization of the ospA protein to the Borrelia cell's outer membrane. Each set of primers also contained sequences near their 5' ends which facilitated cloning of the amplified DNA directly into a high level expression system based on bacteriophage T7 genetic elements. We showed that the full-length OspA protein is synthesized poorly in Escherichia coli and it is associated with the insoluble membrane fraction. In contrast, the truncated form can be expressed to very high levels and it is soluble. The truncated protein was purified to homogeneity and partially characterized. Its N-terminal sequence and molecular weight derived from sodium dodecyl sulfate-polyacrylamide gel electrophoresis agree with those deduced from the DNA sequence. It is a monomer with a native molecular weight of 28,000 and it is very resistant to digestion by trypsin even though it is rather rich in lysine residues (16 mol%). Recombinant OspA protein synthesized in E. coli is recognized by antibodies in sera of Lyme patients, which suggests that the protein may be useful in immunoassays and as a possible immunogen to protect against Lyme borreliosis.     

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Isolation of a tripeptide showing weak acetylthiocholine hydrolysing activity from a soluble form of monkey basal ganglia acetylcholinesterase by limited trypsin digestion

Acetylcholinesterase was purified from the soluble supernatant of monkey (Macaca radiata) brain basal ganglia by a three-step affinity purification procedure. The purified enzyme showed two major protein bands corresponding to molecular weights of approximately 65 kDa and approximately 58 kDa which could be labelled by [3H]diisopropylfluorophosphate. When the purified enzyme was subjected to limited trypsin digestion followed by gel filtration on Sephadex G-75 or Sephadex G-25 column, a peptide fragment of molecular weight approximately 300 Da having a weak acetylthiocholine hydrolysing activity was isolated. The amino acid sequence analysis of this peptide showed a sequence of Gly-Pro-Ser. When the [3H]DFP labelled enzyme was subjected to limited trypsin digestion and Sephadex G-75 column chromatography, a labelled peptide corresponding to approximately 430 Da was isolated. The kinetics, inhibition characteristics and binding characteristics to lectins of this peptide were compared with the parent enzyme. A synthetic peptide of sequence Gly-Pro-Ser was also found to exhibit acetylthiocholine hydrolysing activity. The kinetics and inhibition characteristics of the synthetic peptide were similar to those of the peptide derived from the purified acetylcholinesterase, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 123700 times less than that of the purified AChE.     

The hydrolysis of brain and atrial natriuretic peptides by porcine choroid plexus is attributable to endopeptidase-24.11.

The hydrolysis of the porcine 26-residue brain natriuretic peptide (BNP-26) and its counterpart human 28-residue atrial natriuretic peptide (alpha-hANP) by pig membrane preparations and purified membrane peptidases was studied. When the two peptides were incubated with choroid plexus membranes, the products being analysed by h.p.l.c., alpha-hANP was degraded twice as fast as BNP. The h.p.l.c. profiles of alpha-hANP hydrolysis, in short incubations with choroid plexus membranes, yielded alpha hANP' as the main product, this having been previously shown to be the result of hydrolysis at the Cys7-Phe8 bond. In short incubations this cleavage was inhibited 84% by 1 microM-phosphoramidon, a specific inhibitor of endopeptidase-24.11. BNP-26 was hydrolysed by choroid plexus membranes, kidney microvillar membranes and purified endopeptidase-24.11 in a manner that yielded identical h.p.l.c. profiles. In the presence of phosphoramidon, hydrolysis by the choroid plexus membranes was 94% inhibited. Captopril had no effect and, indeed, no hydrolysis of BNP-26 by peptidyl dipeptidase A (angiotensin-converting enzyme) was observed even after prolonged incubation with the purified enzyme. The stepwise hydrolysis of BNP-26 by endopeptidase-24.11 was investigated by sequencing the peptides produced during incubation. The initial product resulted from hydrolysis at Ser14-Leu15, thereby opening the ring. This product (BNP') was short-lived; further degradation involved hydrolysis at Ile12-Gly13, Arg8-Leu9, Gly17-Leu18, Val22-Leu23, Arg11-Ile12 and Cys4-Phe5. Thus endopeptidase-24.11 is the principal enzyme in renal microvillar and choroid plexus membranes hydrolysing BNP-26 and alpha-hANP.     

Covalent structure of protein C. A second major low molecular weight protein degraded during germination of Bacillus megaterium spores

The complete covalent structure of protein C, a protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 30 and 31 to 71. The intact protein was also cleaved by cyanogen bromide into two peptides, residues 1 to 27 and 28 to 71. Cleavage of the larger cyanogen bromide peptide with trypsin allowed isolation of the COOH-terminal peptide, residues 59 to 71. Automated sequenator analysis of the intact protein and peptide fragments, together with previously published partial sequence data on this protein and carboxypeptidase A digestion of the intact protein provided data from which the following unique sequence was deduced: (formula: see text). The primary sequence of the C protein shows an extremely high degree of homology with that of the A protein--another protein degraded during germination of B. megaterium spores.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.