α_1-抗胰蛋白酶的分离纯化及应用研究进展

α_1-抗胰蛋白酶为人血浆中微量蛋白,其浓缩制剂在临床上具有治疗肺气肿等疾病的确切疗效．综述了近来α_1-抗胰蛋白酶分离纯化工艺和应用研究的进展,结合生化分离纯化技术的发展,讨论了该产品制备新工艺的开发方向．     

人凝血酶原复合物离子交换树脂的筛选及吸附性能研究

采用若干种人工合成的大孔阴离子交换树脂,对人血浆中凝血酶原复合物(PCC)的分离纯化性能进行了研究.同时.将DEAE-Sephadex A50时PCC的分离纯化性能与该类人工合成分离纯化材料进行了对比.研究结果表明,在各种人工合成大孔阴离子交换树脂中,CG-6对PCC具有较优的分离纯化性能;并系统研究了CG-6树脂对PCC分离纯化性能.     

人凝血酶原复合物的研究进展

人凝血酶原复合物(Human prothrombin complex concentrate,PCC)是由健康人混合血浆中分离制备的一种血浆蛋白制剂,主要含有依赖于维生素K的凝血酶原(FⅡ)、凝血酶原转化因子(FⅦ)、抗乙型血友病因子(FⅨ)和自身凝血酶原(FX)。在临床上,PCC主要用于治疗乙型血友病和因维生素K缺乏或患肝病而引起的出血症状。近年来,随着PCC分离技术的发展和安全性的提高,PCC的使用量正在逐年增加。本文综述了PCC的制备工艺研究现状、临床使用和安全性。     

人红细胞膜带3蛋白的提纯与鉴定

提出了一种分离纯化人红细胞膜带3蛋白的不含血型糖蛋白制剂的改良方法:先后用0.89％NaCl、20mM pH8.0磷酸钠和0.05％TritonX-100处理膜除去膜骨骼蛋白类和血型糖蛋白,再用自行设计的凝胶制备电泳装置进一步纯化。冰冻干燥的制剂是均质的,得率为18.5±2.85％,它的分子量、氨基酸组成和紫外吸收光谱与文献报道基本相同。     

超滤技术在生物分离中的应用

“超滤”、“膜分离”和我们日常生活,生产实践息息相关,如安全可用的净化水、血液透析或血液滤过人工肾、发酵产物的分离、牛奶浓缩等。超滤由于成本低．通量高．易于放大的优点,已得到广泛的应用。本文主要介绍了超滤技术在发酵液的澄清（固液分离）、生物产品的精制、药用蛋白的分离纯化以及质粒DNA的分离纯化中的研究进展和应用情况。     

姜老师信箱

蛋白质研讨班学员问:姜老师,您好!我在蛋白质分离纯化方面有两个问题想向您请教:1、工厂规模化生产蛋白(分子量约为15kDa),除了采用各种层析方式外,一般还可以用什么方法来对蛋白质进行浓缩?与实验室规模的蛋白纯化相比是否有不同之处?     

聚球藻PCC 7942氢酶的分离纯化及特性研究

报道了室温、空气环境下聚球藻Synechococcus sp.PCC7942氢酶的分离纯化.经过超声破碎、超速离心、离子交换层析、疏水层析及凝胶层析等步骤,氢酶被纯化了218倍,得率为6.5%,比活为1.46U·mg^-1蛋白.纯化氢酶的SDS-PAGE图显示五条蛋白带,分子量约为83kDa,60kDa,47kDa,30kDa和27kDa.该氢酶为可溶性的双向氢酶,其催化放氢的最佳电子供体为还原态的甲基紫精,最适温度50℃,最适pH8.0.     

Isolation of Plasma and Thylakoid Membranes from the Heterocystous Cyanobacterium Anabaena sp. PCC 7120

具异型胞蓝细菌 Anabaena sp.PCC 7120质膜和类囊体膜的分离纯化 李斌 徐冬一 赵进东*     

重组胰高糖素样肽-1融合蛋白的纯化及其活性测定

主要探讨了发酵液中融合蛋白rh（GLP-1A2G）2-HSA的分离纯化过程。发酵上清液经超滤浓缩、离子交换层析、凝胶过滤分离得高纯度的rh（GLP-1A2G）2-HSA。纯化样品经SDS-PAGE检测为单一条带,HPLC分析纯度达98%,^125I标记后用TCA沉淀法测定放射性纯度达97%,符合药效学和药代动力学研究的需要,整个纯化过程回收率达到48.5%。通过细胞增殖实验表明纯化后的rh（GLP-1A2G）2-HSA具有类似GLP-1的促胰岛β细胞增殖活性,说明该分离过程保护了融合蛋白rh（GLP-1A2G）2-HSA的生物活性。通过融合蛋白rh（GLP-1A2G）2-HSA的纯化方法,可获得高纯度的重组融合蛋白rh（GLP-1A2G）2-HSA,为进一步的药效学和药代动力学研究奠定了基础。     

奶牛Y精子膜蛋白的提取与分析

本实验旨在对奶牛Y精子膜蛋白的提取与SDS-PAGE分析,是分离纯化奶牛Y精子特异膜蛋白基础与关键。本实验采用超声波破碎法将精子膜与精子分离,分离后的精子膜粗品采用蔗糖密度梯度离心法进行纯化。纯化的精子膜经膜蛋白提取液(去污剂)提取,透析浓缩。经SDS-PAGE银盐染色,结果表明该方法成功地提取到膜蛋白,得到膜蛋白电泳图谱。BIO-RAD分析得知Y精子膜蛋白种类至少有10种,分子量范围为7.94kD～166.65kD,其中尤以15kD的蛋白含量最多。奶牛Y精子膜蛋白的提取与鉴定为深入研究Y精子特异膜蛋白在受精过程中的作用,从而获得一种更经济、更安全、更有效的奶牛性别控制方法提供了实验依据。     

Substitution of the Gla domain in factor X with that of protein C impairs its interaction with factor VIIa/tissue factor: lack of comparable effect by similar substitution in factor IX

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.     

Calcium isotope fractionation by intracellular amorphous calcium carbonate (ACC) forming cyanobacteria

The formation of intracellular amorphous calcium carbonate (ACC) by various cyanobacteria is a widespread biomineralization process, yet its mechanism and importance in past and modern environments remain to be fully comprehended. This study explores whether calcium (Ca) isotope fractionation, linked to ACC-forming cyanobacteria, can serve as a reliable tracer for detecting these microorganisms in modern and ancient settings. Accordingly, we measured stable Ca isotope fractionation during Ca uptake by the intracellular ACC-forming cyanobacterium Cyanothece sp. PCC 7425. Our results show that Cyanothece sp. PCC 7425 cells are enriched in lighter Ca isotopes relative to the solution. This finding is consistent with the kinetic isotope effects observed in the Ca isotope fractionation during biogenic carbonate formation by marine calcifying organisms. The Ca isotope composition of Cyanothece sp. PCC 7425 was accurately modeled using a Rayleigh fractionation model, resulting in a Ca isotope fractionation factor (Δ⁴⁴Ca) equal to −0.72 ± 0.05‰. Numerical modeling suggests that Ca uptake by these cyanobacteria is primarily unidirectional, with minimal back reaction observed over the duration of the experiment. Finally, we compared our Δ⁴⁴Ca values with those of other biotic and abiotic carbonates, revealing similarities with organisms that form biogenic calcite. These similarities raise questions about the effectiveness of using the Ca isotope fractionation factor as a univocal tracer of ACC-forming cyanobacteria in the environment. We propose that the use of Δ⁴⁴Ca in combination with other proposed tracers of ACC-forming cyanobacteria such as Ba and Sr isotope fractionation factors and/or elevated Ba/Ca and Sr/Ca ratios may provide a more reliable approach.     

Chromatography in plasma fractionation: benefits and future trends

Industrial-scale chromatographic fractionation and purification methods have been used increasingly in the last few years for plasma fractionation. This has resulted in the development of a new generation of therapeutic plasma derivatives, especially coagulation factors, protease inhibitors and anticoagulants. Implementation and combination of ion-exchange, affinity and size-exclusion chromatography have allowed the development of new therapeutic products with improved purity and safety for treating congenital or acquired plasma protein deficiencies in patients. More recently, the benefit of chromatographic purification of plasma proteins in the removal of plasma-borne viruses has been revealed. Development of packing materials with improved characteristics for industrial applications, including higher capacity and rigidity, should further promote the use of chromatography as an essential plasma fractionation tool and confine more and more the traditional ethanol precipitation methods to the final processing stages used to recover albumin.     

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.     

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin‐enriched fractions based on polymer‐salt aqueous two phase system (ATPS). A small‐scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50‐fold scale‐up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75‐fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1005–1011, 2012     

The N-terminal epidermal growth factor-like domain of coagulation factor IX. Probing its functions in the activation of factor IX and factor X with a monoclonal antibody

The absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor-like (EGF) domains and a serine protease domain. In this study, the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined, and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by activated factor XI, but activation by activated factor FVII-tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent k(cat) for factor X both in the presence and absence of activated factor VIII. Based on these results, we produced a preliminary model of the structure of the activated factor IX-activated factor VIII-AW complex on the surface of phospholipid. The model suggests that in the Xase complex, EGF1 of activated factor IX is not involved in direct binding to activated factor VIII. Studies of the interaction of antibody AW with a mutated FIX molecule (R94D) also suggest that the Glu(78)-Arg(94) salt bridge is not important for maintaining the structure of FIX.     

Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function

Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects.     

昆虫谷胱甘肽S-转移酶蛋白纯化技术研究

昆虫谷胱甘肽S-转移酶蛋白纯化的主要方法是沉淀法、层析法和电泳法。沉淀法是在进行柱层析前常使用的一种方法,但是其纯化倍数较低。层析法纯化倍数较高,但其费用也较高。电泳通常是作为一种辅助方法来使用。利用这些方法,谷胱甘肽S-转移酶蛋白最高纯化倍数为1138倍(马素永等利用层析法和电泳法对亚洲玉米螟谷胱甘肽S-转移酶蛋白进行纯化)。文章讨论了昆虫谷胱甘肽S-转移酶蛋白纯化研究的应用。     

Ca(2+) -induced binding of anticoagulation factor II from the venom of Agkistrodon acutus with factor IX

Anticoagulation factor II (ACF II), a coagulation factor X- binding protein from the venom of Agkistrodon acutus has both anticoagulant and hypotensive activities. Previous studies show that ACF II binds specifically with activated factor X (FXa) in a Ca(2+) -dependent manner and inhibits intrinsic coagulation pathway. In this study, the inhibition of extrinsic coagulation pathway by ACF II was measured in vivo by prothrombin time assay and the binding of ACF II to factor IX (FIX) was investigated by native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR). The results indicate that ACF II also inhibits extrinsic coagulation pathway, but does not inhibit thrombin activity. ACF II also binds with FIX with high binding affinity in a Ca(2+) -dependent manner and their maximal binding occurs at about 0.1 mM Ca(2+) . ACF II has similar binding affinity to FIX and FX as determined by SPR. Ca(2+) has a slight effect on the secondary structure of FIX as determined by circular dichroism spectroscopy. Ca(2+) ions are required to maintain in vivo function of FIX Gla domain for its recognition of ACF II. However, Ca(2+) at high concentrations (>0.1 mM) inhibits the binding of ACF II to FIX. Ca(2+) functions as a switch for the binding between ACF II and FIX. ACF II extends activated partial thromboplastin time more strongly than prothrombin time, suggesting that the binding of ACF II with FIX may play a dominant role in the anticoagulation of ACF II in vivo.