啤酒酵母生产的重组水蛭素的纯化及脱色

对啤酒酵母生产的重组水蛭素变异体1(rHV1)进行多步骤的纯化。首先将分泌到培养上清液中的水蛭素进行硫酸铵沉淀和SephadexG-50凝胶过滤,再用Q-SepharoseHP阴离子交换层析分离,最后用HPLCSP-5PW阳离子交换柱脱色及HPLCC8柱反相层析。真空干燥后得到的水蛭素蛋白经SPS-PAGE、N端氨基酸序列分析、抗凝血酶活力分析鉴定,证明已获得高纯度的重组水蛭素HV1制剂,为利用基因工程方法生产重组水蛭素的规模化生产及临床应用提供了依据     

Coexpression of BiP increased antithrombotic hirudin production in recombinant Saccharomyces cerevisiae

In order to increase a production level of antithrombotic hirudin, BiP was simultaneously expressed in recombinant Saccharomyces cerevisiae strains carrying ten and 15 copies of the hirudin expression cassette integrated in the chromosome. Coexpression of BiP greatly enhanced both cell growth and hirudin production in recombinant S. cerevisiae. Maximum hirudin concentration of 36 mg l(-1) was obtained from batch culture of the ten copy-number transformant concomitantly harboring an episomal copy of the BiP gene under the control of the GAL1 promoter, which is corresponding to a 2.5-fold increase compared with the control strain carrying the genomic BiP gene only. The mean size of the recombinant yeast cells expressing the BiP gene remained at a relatively constant level compared with the control strains of which size increased after the onset of hirudin expression by the GAL10 promoter.     

人组织型纤溶酶原激活剂-水蛭素融合基因的构建及其在毕赤酵母中的表达

构建并表达兼有溶栓和抗凝活性、减少出血副作用的人组织型纤溶酶原激活剂(t-PA)和水蛭素(HV2)的融合蛋白。通过提取总RNA和RT-PCR获得t-PA基因,与HV2基因通过活化凝血因子X(Fxa)识别序列（IEGR）的对应碱基序列连接构成融合蛋白基因,融合蛋白基因经pGEM-T、pIC9克隆至表达载体pIC9K上,电转导入毕赤酵母（Pichia pastoris）GS115。转化子摇瓶内甲醇诱导表达。纤维蛋白平板溶圈法和纤维蛋白凝块法分别检测溶栓和抗凝活性。琼脂糖凝胶电泳结果显示克隆的t-PA基因片段大小为1700bp,序列测定结果表明其35位氨基酸由文献报道的精氨酸突变为色氨酸。限制性酶切和PCR鉴定结果均表明融合蛋白基因已克隆入表达载体和宿主菌。甲醇利用实验、G418抗性筛选获得多拷贝甲醇利用快型克隆。甲醇诱导表达产物具有纤溶活性并可被抗t-PA抗体抑制。完整融合蛋白无抗凝活性,但以Fxa裂解后可释放抗凝活性。同时,融合蛋白以单链和双链两种形式存在。融合蛋白在血栓部位特有的Fxa作用下靶向释放抗凝活性,具有溶栓抗凝双功能,有望降低临床出血副作用。     

Rapid purification and revised amino-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech

Hirudin is a specific polypeptide thrombin inhibitor consisting of 65 amino acids that is produced by the leech, Hirudo medicinalis. We describe a rapid method for the purification of hirudin from a leech extract. Crude hirudin, purchased from a commercial source, was first fractionated on a DEAE-HPLC column using a salt gradient. Hirudin activity was monitored by inhibition of the thrombin-mediated hydrolysis of a synthetic substrate H-D-Phenylalanyl-Pipecolyl-Arginine-p-Nitroanilide. The fractions containing antithrombin activity were pooled and further purified by reverse-phase HPLC. The homogeneity of purified hirudin was confirmed by a single amino-terminal sequence for 43 residues with Val-Val as the first two amino acids. Residue 33 was Asn rather than Asp as reported previously.     

Thrombin-induced thromboxane generation by neutrophils and lymphocytes: dependence on enzymic site

We examined the effects of thrombin on thromboxane generation by sheep neutrophils and lymphocytes in vitro. Physiological concentration of thrombin (50 nM) resulted in thromboxane B2 generation from both neutrophils and lymphocytes, which was comparable to that obtained with zymosan activated serum challenge of the cells. Thromboxane B2 generation was dependent on the enzymic region of the thrombin molecule responsible for clotting activity because the complexing of thrombin with hirudin (1:1 U:U mixture of thrombin and hirudin) abolished thromboxane generation from both cell types. Further studies with modified forms of alpha-thrombin (which were produced by irreversible conjugation at the catalytic site and lacked enzymic activity) also showed no generation of thromboxane B2 from neutrophils or lymphocytes. The results indicate that thrombin stimulates thromboxane generation from neutrophils and lymphocytes and that this response is dependent on the proteolytic activity of thrombin.     

Deciphering the structural elements of hirudin C-terminal peptide that bind to the fibrinogen recognition site of alpha-thrombin

The C-terminal peptide of a hirudin acts as an anticoagulant by binding specifically to a noncatalytic (fibrinogen recognition) site of thrombin. This binding has been shown to shield five spatially distant lysines of the thrombin B-chain (Lys21, Lys65, Lys77, Lys106, and Lys107). It was also demonstrated that modification of the sequence of the hirudin C-terminal peptide invariably diminished its anticoagulant activity. The major object of this study is to investigate how the decreased activity of the modified hirudin C-terminal peptide is reflected by the change of its binding properties to these five lysines of thrombin. A synthetic peptide representing the last 12 C-terminal amino acids of hirudin (Hir54-65) was (1) truncated from both its N-terminal and its C-terminal ends, or (2) substituted with Gly along residues 57-62, or (3) chemically modified to add (sulfation at Tyr63) or abolish (Asp and Glu modification with carbodiimide/glycinamide) its negatively charged side chains. The binding characteristics of these peptides to thrombin were investigated by chemical methods, and their corresponding anticoagulant activities were studied. Our results demonstrated the following: (1) the anticoagulant activities of hirudin C-terminal peptides were quantitatively related to their abilities to shield the five identified lysines of thrombin. The most potent peptide was sulfated Hir54-65 (S-Hir54-65) with an average binding affinity to the five lysines of 120 nM. A heptapeptide (Hir54-60) also displayed anticoagulant activity and thrombin binding ability at micromolar concentrations. (2) All active hirudin C-terminal peptides regardless of their sizes and potencies were shown to be capable of shielding the five lysines of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin

The hirudin variant HV2 was modified by in vitro site-specific mutagenesis of HV2 cDNA to generate HV2(Asn-47----Lys), HV2(Asn-47----Arg) and HV2(Lys-35----Thr, Asn-47----Lys). Residues 35 and 47 are positioned respectively within the finger and prothrombin-like domains of hirudin, both of which have been suggested as thrombin binding sites. The modified polypeptides were synthesized in Saccharomyces cerevisiae using a secretion vector and purified from culture supernatants. By analysis of the human alpha-thrombin:hirudin inhibition reaction in steady-state conditions it was shown that the dissociation constants for HV2(Lys-47) and HV2(Arg-47) were 5- to 14-fold lower than for unmodified HV2, whereas mutation of Lys-35 did not significantly alter the inhibition kinetics. Furthermore, HV2(Lys-47), whose sequence is identical to a natural hirudin variant, displayed enhanced anti-thrombotic activity in vivo, having a 100-fold lower ED50 compared to HV2 in the rabbit Wessler venous thrombosis model. These results support a role for the prothrombin-like domain in thrombin binding and, moreover, demonstrate that in vivo antithrombotic efficiency correlates with the dissociation constant of the inhibition reaction.     

Hirudin variants production by genetic engineered microbial factory

Hirudin was discovered as an active anticoagulant in leech extracts almost 60 years ago. Since their initial discovery, hirudin and its variants have been produced with various anti-thrombotic, cancer cell inhibition, diabetic cataract treatment and anti-fatigue activities. Some hirudin variants have been approved for clinical use and released into the marketplace. Recent progress has seen made in relation to hirudin variants expressed in several well-established microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and others, with high levels of activity and yield. This review summarizes the current progress on hirudin production using microbial producers, and considers the outlook for future development.     

The structure of the yeast ribosomal RNA genes. 4. Complete sequence of the 25 S rRNA gene from Saccharomyces cerevisae.

The complete nucleotide sequence of the 25 S rRNA gene from one rDNA repeating unit of Saccharomyces cerevisiae has been determined. The corresponding 25 S rRNA molecule contains 3392 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) or 1.17 x 10(6). Striking sequence homology is observed with known 5'- and 3'-end terminal segments of L-rRNA from other eukaryotes. Possible models of interaction with 5.8 S rRNA are discussed.     

Note : Genetic and biochemical characterization of nisin Z produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719

The bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719 was purified and characterized. Two peaks exhibiting antimicrobial activity were obtained after purification. Primary structure of the peptide of major peak 2 was identical to that of nisin Z when determined by Edman degradation and confirmed by DNA sequence analysis. The molecular mass as determined by mass spectrometry was 3346·39 ± 0·40 Da for peak 1 and 3330·39 ± 0·27 Da for peak 2, which suggests that peak 1 may correspond to an oxidized form of nisin Z. The two purified peaks exhibiting xrantimicrobial activity appear to correspond with the oxidized and native forms of nisin Z.     

Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.     

Construction and characterization of trifunctional single-chain urokinase-type plasminogen activators.

Two chimeric proteins have been constructed. One consists of four parts: a portion of the low molecular mass single-chain urokinase-type plasminogen activator (scu-PA-32K, residues 144-411), a 15-mer linker sequence, the C-terminal amino-acid sequence (residues 53-65) of hirudin (Hir), and an RGD sequence derived from the leech protein decorsin, i.e. scu-PA(32 k)-linker-Hir (residues 53-65)-RGD peptide. The other comprises two main segments: scu-PA(32 k) and hirudin into which RGDSP is inserted between its residues 33 and 34, i.e. hirudin (residues 1-33)-RGDSP-hirudin (residues 34-65)-scu-PA(32 k). These two chimeric genes were expressed in Escherichia coli, and the products were purified by Zn2+-chelating Sepharose 4B chromatography and benzamidine Sepharose 6B chromatography. Our results suggested that these two chimeric proteins not only had plasminogen-dependent fibrinolytic activity, but also possessed platelet aggregation inhibitory activity and antithrombin activity.     

High-level production of recombinant fungal endo-beta-1,4-xylanase in the methylotrophic yeast Pichia pastoris

Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.     

Engineering of a staphylokinase-based fibrinolytic agent with antithrombotic activity and targeting capability toward thrombin-rich fibrin and plasma clots

Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.     

Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal delta-integration

Recombinant Saccharomyces cerevisiae strains were developed to overproduce an anticoagulant hirudin. The delta-sequences of the yeast retrotransposon Ty1 and URA3 were used as target sites for a hirudin expression cassette. High copy-number transformants were successfully selected using a dominant selection antibiotic, G418. The copy numbers of the hirudin expression cassette integrated into delta-sequences of the yeast chromosome ranged from five to ten copies per cell. Production of hirudin in the delta-integrated recombinant S. cerevisiae system increased over two-fold compared with the YEp-based episomal hirudin expression system. A linear relationship between the copy number of the hirudin expression cassette and hirudin expression level was observed up to 10 copies. The hirudin expression cassettes integrated into the yeast chromosome were stably maintained in non-selective culture conditions.     

Purification and properties of an esterase from the yeast Saccharomyces cerevisiae and identification of the encoding gene.

We purified an intracellular esterase that can function as an S-formylglutathione hydrolase from the yeast Saccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50 degrees C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to S-formylglutathione by S. cerevisiae.     

Purification and identification of a human dermal extract component inhibitory to fibroblast proliferation.

It was previously shown that a citric acid buffer extract of human dermis (extract D) inhibited growth of human diploid fibroblasts in monolayer culture (Muir et al., 1997). Further fractionation has shown that the active principle is probably a proteoglycan, and that retention of its inhibitory activity is dependent on the use protease inhibitors throughout the extraction procedure. Elution of extract D from a DEAE-cellulose column produced four major peaks, each of which was subjected to SDS-PAGE as well as being tested for inhibitory activity on the growth of fibroblasts in culture. Peaks III and IV had no inhibitory effect, but peak I contained highly active material. Gels of this peak showed prominent bands of 120 kDa (corresponding to dermatan sulphate proteoglycan II, DS-PG II) and at 45 kDa (corresponding to the core protein). The latter band became more prominent when extract D which had been treated with chrondroitinase ABC was electrophoresed. Their identities were verified by Western blotting. Peak II also contained some slower-acting inhibitory material which has as yet to be identified, but contains little or no protein corresponding to the decorin core-protein. The data indicate that the intact decorin molecule, DS-PG II, is the main inhibitory principle in human skin.     

Bovine amyloid protein AA: isolation and amino acid sequence analysis

Amyloid-laden renal glomeruli were selectively isolated from a cow with a history of multiple organ inflammatory diseases which terminated in amyloid-induced glomerulopathy and severe proteinuria. Lyophilized amyloid fibrils obtained by water extraction procedures were dissolved in 6M guanidine hydrochloride and gel filtered on Sepharose CL6B and Sephacryl S-300 Superfine columns for slab gel electrophoresis, analytic isoelectric focusing, and amino acid sequence analyses. Electrophoresis of material from the major retarded peak of the elution profile revealed that bovine protein AA moves as one band with an apparent molecular mass of about 14,000 Daltons. Several distinct bands between approximately pH 4.0 and 5.0 were observed when this material was evaluated by analytic isoelectric focusing, thus having a pattern resembling that of human and dog protein AA. A blocked N-terminus was demonstrated when protein from the major retarded peak was subjected to amino acid sequencing, but cyanogen bromide cleavage followed by gel filtration produced 3 peptide fragments for amino acid sequence analysis. These peptides had a high degree of homology with positions 4-14, 18-24 and 25-49 of human protein AA. An apparent complete homology between bovine protein AA and protein AA from other species was apparent at positions 35-45, providing further evidence that this is a functionally significant part of the serum protein AA (SAA) molecule.     

Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.