重组水蛭素HV2的稳定性

重组水蛭素ＨＶ２是凝血酶的特异性抑制剂,是一种非常稳定的蛋白质,温度的升高（１００℃水浴）和ＰＨ（１－１３）的改变不影响其活力,在某些变性剂（８ｍｏｌ／Ｌ素、１％ＳＤＳ和６ｍｏｌ／Ｌ盐酸胍）存在的条件下也非常稳定,０．１ｍｏｌ／Ｌ的ＤＴＴ在７０℃时使其部分失活,只有ＰＨ和温度同时升高其活力才开始下降,ＰＨ１３、８０℃处理１５ｍｉｎ即完全失活,氨基酸组成和活性分析发现失活样品的Ｃｙｓ和Ｌｙｓ被破坏。     

重组水蛭素HV2的稳定性

重组水蛭素ＨＶ２是凝血酶的特异性抑制剂,是一种非常稳定的蛋白质。温度的升高（１００℃水浴）和ｐＨ（１─１３）的改变不影响其活力,在某些变性剂（８ｍｏｌ／Ｌ尿素、１％ＳＤＳ和６ｍｏｌ／Ｌ盐酸胍）存在的条件下也非常稳定,０．１ｍｏｌ／Ｌ的ＤＴＴ在７０℃时使其部分失活,只有ｐＨ和温度同时升高其活力才开始下降,ｐＨ１３、８０℃处理１５ｍｉｎ即完全失活,氨基酸组成和活性分析发现失活样品的Ｃｙｓ和Ｌｙｓ被破坏。重组水蛭素ＨＶ２含有一个结构紧密的Ｎ端核心区和一个无序的Ｃ端尾部。其Ｎ端的３个Ｌｙｓ－Ｘａａ键均不被胰蛋白酶水解;胃蛋白酶及糜蛋白酶消化后,分离所得片段,氨基酸组成分析发现Ｎ端核心区依然保持很高的抗凝血酶活性,继续消化２４ｈ,核心区不被进一步降解。     

重组水蛭素的纯化和鉴定

本文报导了化学合成的水蛭素基因在酵母细胞中得到表达,井能分泌水蛭素到胞外。将该菌株培养物的上清液经硫酸铵沉淀和Sephadex G-50过滤后,用DEAE-SephadexA-25进行阴离子交换层析,进而用HPLC反相层析,得到表达产物重组水蛭素。经SDS-PAGE,氨基酸序列分析,抗凝血酶活力分析及血浆滴定实验等方法鉴定,证明该基因表达产物与天然水蛭素HV_2相同。     

重组双功能水蛭素质量标准研究

以生色底物法测定抗凝血酶活性,比浊法测定抗血小板聚集活性,还原型SDS-PAGE测定分子量,SDS-PAGE和反相高效液相色谱测定纯度,毛细管电泳法测定等电点,胰蛋白酶酶切后进行肽图分析,其余检测项目按《中国药典》2005版三部规定进行。结果显示,用建立的方法对原液和成品进行了检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》2005版三部的要求。建立的质控方法和质量标准具有保证产品安全、有效、质量可控的特点,可用于重组双功能水蛭素产品的常规检定。     

抗凝良药水蛭素的研究进展

从水蛭素的分子生物学性质,克隆表达研究,以及临床应用研究等主要方面论述抗凝防栓良药水蛭素的研究进展,并对水蛭的临床应用价值,开发研究成为抗栓领域的一大热点进行了论述。     

毕赤酵母发酵液的脱色和重组水蛭素的分离

用毕赤酵母表达重组水蛭素的发酵液中含有大量色素和一些杂蛋白。本文对发酵上清的脱色和重组水蛭素的分离进行了探讨。鉴于水蛭素的稳定性 ,用加热法预处理发酵上清取得了满意的效果。对离子交换、疏水层析、凝胶过滤、羟基磷灰石吸附层析以及它们的优化组合进行了研究 ,结果表明 ,阳离子交换层析、凝胶过滤和阴离子交换层析的组合对发酵上清的脱色和重组水蛭素的分离是有效的。     

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     

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.     

The pharmacology of recombinant hirudin

A review about comprehensive studies on pharmacological properties of hirudin produced by genetic engineering is given. Anticoagulant effects, influence on platelet functions, studies on toxicity and side effects as well as the pharmacokinetic behaviour and antithrombotic actions of this highly effective thrombin inhibitor are described.     

Effects of methanol on expression of an anticoagulant hirudin in recombinant Hansenula polymorpha

A series of batch, fed-batch, and continuous cultures was carried out to analyze the effects of methanol on the fermentation characteristics of recombinant Hansenula polymorpha for the production of hirudin, an anticoagulant. Hirudin expression efficiencies were greatly influenced by the methanol concentrations in continuous and fed-batch culture modes. At a steady state of continuous culture, an optimum methanol concentration of 1.7 g l⁻¹ was determined at a dilution rate of 0.18 h⁻¹ with 1.8 mg l⁻¹ h⁻¹ hirudin productivity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 58–61. Received 21 September 2000/ Accepted in revised form 10 June 2001     

Characterization of C-terminal-truncated urinary metabolites of a recombinant hirudin in rats

Although it has been reported that hirudin was excreted in urine mainly as its nonmetabolized form in humans, dogs, and rabbits, no report has been published about the molecular nature of urinary metabolites in rats. We found that nonmetabolized hirudin could not be detected in rat urine after its i.v. administration and that urinary metabolites of recombinant hirudin CX-397 consisted of at least the following six C-terminal-truncated peptides: CX-397^1–49, CX-397^1–50, CX-397^1–51, CX-397^1–52, CX-397^1–54, and CX-397^1–55, in the ratio of roughly 11, 51, 3, 11, 19, and 5%, respectively. In conclusion, the urinary metabolism of recombinant hirudin in rats is different from that in humans, dogs, and rabbits, suggesting that the handling of hirudin in rat kidney is unique among them.     

Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Hirudin can be used as an oral anticoagulant and antithrombotic agent. The hirudin variant III gene, derived from the medicinal leech, Hirudo medicinalis, was fused to SP310mut2 signal sequence and expressed by a nisin-controlled gene expression system in Lactococcus lactis which was then grown in a 7 l fermenter. After induction with 8 ng nisin ml⁻¹, the product was secreted into the culture medium and accumulated up to ~2.7 mg l⁻¹. MALDI-TOF/MS and anticoagulant activity analyses on the purified product confirmed its authenticity. This is the first demonstration that hirudin can be extracellularly secreted and correctly processed in L. lactis.     

Generation of bioactive recombinant Ancylostoma caninum anticoagulant peptide c2

Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction. Recently, several anticoagulant serine protease inhibitors have been identified from adult Ancylostoma caninum hookworms. One of them, A. caninum anticoagulant peptide c2 (AcAPc2), can inhibit the activity of factor VIIa/tissue factor complex to exert its antithrombotic effect. However, it is difficult to adopt traditional expression and purification systems to yield high-purity recombinant AcAPc2 (rAcAPc2). Here, we employed a simple method to produce high-yield and high-purity rAcAPc2. We obtained the full-length double-stranded cDNA encoding AcAPc2 by overlapping PCR and cloned it into an intein-based expression vector. The AcAPc2 cDNA was expressed in Escherichia coli and comprised 30% of the total bacterial proteins. The expressed rAcAPc2 was purified by cleaving the fused chitin-binding domain at pH 7.2. Finally, we produced a high yield of rAcAPc2 at a purity of greater than 98%. Importantly, the generated rAcAPc2 prolonged the prothrombin time (PT) and activated partial thromboplastin time (aPTT) of human plasma in vitro in a dose-dependent manner. Therefore, this method to generate the high-purity and bioactive rAcAPc2 may contribute to the scientific research on its biological function and the treatment of thrombotic diseases.     

Suppression of proteolytic degradation of recombinant hirudin from Saccharomyces cerevisiae using the O2-enriched air

A novel process using O2-enriched air supply was used to suppress the C-terminal proteolytic degradation of recombinant hirudin (r-hirudin) from Saccharomyces cerevisiae. When dissolved O2 was controlled above 20% saturation level using normal air, inactive forms of C-terminally truncated hirudin were observed in culture broth from 48 h of fermentation. The use of O2-enriched air giving above 40% saturation of dissolved O2 suppressed the proteolytic degradation and hence the formation of truncated forms of inactive r-hirudin until 60 h of fermentation.     

The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.     

Resistance to the anorexic and thermogenic effects of centrally administrated leptin in obese aged rats

The aim of the present study was to determine whether the anorexic and thermogenic effects of leptin were attenuated in overweight aged rats following intracerebroventricular (i.c.v.) injection of murine leptin. Male F344/BN rats of two ages (6 months: young (n=20) and 24 months: old (n=18)) were divided into three groups (control, pair-fed and leptin) and were treated with either vehicle (artificial cerebrospinal fluid) or leptin (15.6 microgram/day) for 3 days. There was an age-related increase in basal food intake (20+/-2%), serum leptin levels (363+/-106%) and leptin (OB) mRNA (72+/-16%) in perirenal white adipose tissue (PWAT). In contrast, basal expression of hypothalamic NPY mRNA and brown adipose tissue (BAT) uncoupling protein 1 (UCP1) mRNA was reduced significantly (-35+/-4% and -51+/-5%, respectively) with age. I.c.v. leptin treatment had a significantly greater effect in reducing food intake (-42+/-5% vs. -23+/-4%), serum leptin levels (-55+/-7% vs. 10+/-2%) and PWAT OB mRNA (-46+/-2% vs. 10+/-5%) in young than in old rats. Similarly, central leptin treatment also had a greater effect in suppressing hypothalamic NPY mRNA expression in young (-23+/-4%) than in old (-8+/-4%) rats compared with their age-matched pair-fed treated rats. The stimulatory effect of i.c.v. leptin treatment on BAT UCP1 mRNA expression was also significantly greater in young rats (45+/-8%) than in old rats (10+/-6%) compared with age-matched pair-fed rats. Our previous report indicated that these overweight aged rats were resistant to peripheral administered leptin. The present data extend those findings and demonstrate that the impaired anorexic and metabolic effects of leptin are centrally mediated. This leptin resistance may be due to either the elevated obesity and serum leptin with age or due to age itself or both. The development of leptin resistance with age may contribute to the hyperphagia, hyperleptinemia and impaired energy balance with age.