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1.
天花粉蛋白Y14F/R22L定点突变及其活性研究   总被引:1,自引:0,他引:1  
利用多聚酶链式反应(PCR)技术,对天然天花粉蛋白(nTCS)基因在Tyr14和Arg22两个保守残基处同时进行定点突变,即Tyr14变成Phe,Arg22变成Leu,然后克隆到pET-8c高效表达载体上,构建成重组质粒pETY14F/R22L.经序列分析,定点突变的结果与预先设计的完全一致,突变后的天花粉蛋白命名为Y14F/R22LTCS.将pETY14F/R22L转化到E.coliBL21(DE3,pLysS)中,进行表达.经CM-SepharoseCL-6B柱纯化,SDS-PAGE鉴定,纯度可达90%.RIP活性测定显示,Y14F/R22LTCS的活性比nTCS降低了7.5倍,活性变化不显著,因此,TCS的Try14和Arg22对维持其活性部位构象并不是必需的.但由于Y14F/R22LTCS在E.coli中的表达量与nTCS相比明显下降,因此,Tyr14和Arg22可能与TCS翻译后的折叠有关.  相似文献   

2.
人肝癌细胞株7721细胞的N-乙酰氨基葡萄糖转移酶Ⅲ(GnTⅢ)活性受Ser/Thr蛋白激酶的两种抑制剂quercetin和三氟吡嗪(TFP).蛋白激酶C(PKC)的两种特异性抑制剂D-鞘氨醇和staurosporine的抑制。用PMA处理细胞舌,GnTⅢ活力随膜性PKC(m-PKC)活力而平行变化,但与胞液PKC活力的变化无关。Quercetin、D-鞘氨醇和staurosporine还能够阻断PMA对GnTⅢ的激活。Quercetin、staurosporine对m-PKC和GnTⅢ的抑制作用基本上与它们的应用浓度成正比关系。当人及大鼠肾脏的粗GnT制剂分别用碱性磷酸酶切除磷酸基后,GDTⅢ的活力明显下降。这些结果表明m-PKC可能通过蛋白质的Ser/Thr残基上磷酸化和去磷酸化作用直接或间接地调节GnTⅢ。  相似文献   

3.
青霉素酰化酶活性中心的定点突变   总被引:7,自引:1,他引:6  
对E.cole ATCC 11105的青霉素酰化酶(penicillin G acylase,PGA)活性中心的Ser290分别进行了定点突变和和化学修饰,将活性中心的Ser290改变成Cys或Secys,PGA水解活力均大为下降,但仍保留部分活性。其对底物6-硝基(3-苯乙酰氨基)苯甲酸(6-nitro-3-phenylacetamido benzoic acid,NIPAB)的kcat分别从1  相似文献   

4.
采用8-(6-氨己基)-氨基-5'-AMPSepharose亲和层析法和DEAE-Sepharose离子交换层析法从大熊猫心肌中分离纯化出了乳酸脱氢酶同工酶H4.纯化的大熊猫LDH-H4,比活为445U/mg蛋白,经SDS-PAGE,PAGE,等电聚焦电泳鉴定均为一条带,其亚基分子量为36000,等电点为5.45.经测定大熊猫LDH-H亚基N端被封闭,C端氨基酸残基经测定为Leu.氨基酸组成分析表明每个亚基含有5个Cys,9个Met.  相似文献   

5.
在人肝癌细胞7721中研究了酪氨酸蛋白激酶(TPK)和蛋白激酶C(PKC)的激活剂[分别为表皮生长因子(EGF)和佛波酯(PMA)]和各种蛋白激酶抑制剂对N-乙酰氨基葡萄糖转移酶V(GnT-V)活力的影响,以探讨TPK和PKC对GnT-V的调节。结果发现,EGF或PMA处理细胞48h后,GnT-V的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮(genistein)在抑制TPK和PKC的同时,抑制GnT-V的基础活力,并完全阻断EGF或PMA对GnT-V的增高作用;TPK的特异性抑制剂Tyrphostin-25和PKC的特异性抑制剂D-鞘氨醇分别应用时,各自只能部分地取消EGF或PMA对GnT-V的诱导。但当Tyrphostin-25和D-鞘氨醇同时加入培养基中则可完全阻断EGF或PMA对GnT-V的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制GnT-V的基础活力,也可完全消除EGF或PMA对GnT-V的激活。以上结果提示EGF或PMA通过蛋白激酶调节GnT-V的酶蛋白合成,并且GnT-V受到膜性TPK和PKC的双重调节,其中m-TPK较m-PKC更为重要。  相似文献   

6.
家蚕滞育生物钟蛋白质Ease A4的纯化及其分子结构分析   总被引:2,自引:0,他引:2  
EaseA4是家蚕卵的一种滞育生物钟蛋白质.产下后2d的家蚕C108品种滞育性卵,经过丙酮脱脂、85℃热处理、硫酸铵沉淀和SephadexG-25凝胶过滤层析初步分离,进一步经过Sep-PakC18脱盐浓缩,HPLC(柱为YMC-PackProtein-RP)分离,通过SDS-PAGE和MALDIMS方法鉴定,纯化得到EaseA4蛋白质.从10g蚕卵最终得到了11.8μgEaseA4.EaseA4由从His到Tyr的155个氨基酸残基构成,蛋白质部分的分子量为16601.其22位氨基酸残基Asn处有一个Asn-X-Thr糖基化场所,并有糖基结合在该部位,糖基的分子量约为760.EaseA4的61位和150位的两个Cys氨基酸残基之间存在二硫键.糖基和二硫键的存在不仅有利于酶蛋白的分离,还可能与酶活性有关  相似文献   

7.
用浸泡法得到了(E160A)天花粉蛋白(trichosanthin, TCS),(E160D)TCS与Ade 和(E160A)TCS与FMP复合物的晶体.在Mar Research 面探测器系统上分别收集了0.20 nm ,0.19nm 和0.205 nm 分辨率的X 射线衍射数据,数据处理用Mar Scale 程序系统完成.用同晶差值Fourier法解析了(E160A)TCS-Ade,(E160D)TCS-Ade 和(E160A)TCS-FMP的晶体结构,结构修正利用X-PLOR程序,修正结果,晶体学R因子分别为0.166,0.176,0.179.键长和键角的RMS偏差分别为0.0010 nm 和2.503°,0.0013 nm 和2.665°,0.0012 nm 和2.676°.在这三个结构中均未见到Glu189侧链方向的改变.Ade 或FMP仍结合在N-糖苷酶活性口袋之中,它夹在Tyr70和Tyr111两个侧链环之间,与Tyr70环近乎平行.这一结果表明:TCS中的Glu160分别突变成Ala 和Asp,仍能与AMP发生N-糖苷酶反应,但是活性降低了一些.可见Glu160对TCS与AMP的作用是重要的,但不是必要的.  相似文献   

8.
日本血吸虫26kD抗原基因在BCG中的表达   总被引:5,自引:0,他引:5  
研究了外源基因日本血吸虫26kD抗原(Sj26GST)在卡介苗(bacilusCalmete-Guerin,BCG)、耻垢分枝杆菌(M.smegmatis)和大肠杆菌(E.coli)中的表达.运用重组DNA和聚合酶链反应(PCR)等分子生物学技术,以表达Sj26GST的E.colipGEX衍生质粒为模板,经PCR得到编码Sj26GST的全长cDNA片段.将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白(heatshockprotein,HSP)70的启动子下游,再将HSP70启动子和Sj26GST基因一起亚克隆到E.coli-分枝杆菌穿梭质粒pBCG-2000中,得到E.coli-分枝杆菌穿梭表达质粒pBCG-Sj26.pBCG-Sj26电转化入BCG和M.smegmatismc2155中表达Sj26GST抗原,所表达的天然重组Sj26GST(rSj26GST)为可溶性蛋白,在SDS-PAGE上分子量为26kD处可见明显的表达蛋白带.其表达量分别占BCG和M.smegmatis菌体总蛋白的15%和10%.可见,Sj26GST基因能在BCG中高效表达.  相似文献   

9.
蛋白质定点PEG化研究:97Cys-IFH-γ的巯基专—PEG修饰唐微,常远,徐 飞,郑仲承,刘新垣(中国科学院上海生物化学研究所,200031)关键词人干扰素-γ;定点修饰;半胱氨酸目前进行的PEG化反应,多是针对蛋白质肽链上自由末端氨基进行的,由...  相似文献   

10.
通过培养的人主动脉平滑肌细胞(hASMC)及脐静脉内皮细胞(hUVEC),应用3H-TdR参入、Northernblot分析、逆转录多聚酶链反应(RT-PCR)、放射免疫分析(RIA)、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖(HSPG)对hASMC和hUVECDNA合成的作用及对血小板源生长因子(PDGF)、PDGF受体、转化生长因子β(TGF-β)、内皮素-1(ET-1)或碱性成纤维细胞生长因子(bFGF)基因表达和肾素-血管紧张系统(RAS)的影响,结果显示,HSPG明显抑制培养的hASMC基础的DNA合成(cpm值为:10385±3263vs,25541±6421,P<0.01)及外源性PDGF诱导的DNA合成(cpm值为:9878±1947vs.13481±44l0,P<0.05);抑制PDGFA链、TGF-Bp和ET-1mRNA表达,提高PDGFa和β受体mRNA的表达;显著降低hASMC培养液中血管紧张素Ⅱ(AngⅡ)的浓度和血管紧张素转换酶(ACE)的活性,推测HSPG抑制PDGFA链、TGF-β及ET-1mRNA表达,降低ACE活性及AngⅡ浓度是其抑制hASMC增殖的重要机  相似文献   

11.
He XH  Shaw PC  Xu LH  Tam SC 《Life sciences》1999,64(14):1163-1175
Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.  相似文献   

12.
X H He  P C Shaw  S C Tam 《Life sciences》1999,65(4):355-368
PEG modification (PEGylation) has been shown to reduce immunogenicity and prolong circulating half-life of proteins. In the present study, site-directed PEGylation was used to reduce immunogenicity and prolong plasma half-life of trichosanthin (TCS). Four TCS mutants, i.e. S7C, Q219C, K173C and [K173C,Q219C] (KQ), were constructed by site-directed mutagenesis. PEG modifications were done by reacting PEG5k-maleimide or PEG20k-maleimide reagent with the newly introduced cysteine residue of the mutants. The plasma clearance rate of PEGylated TCS mutants decreased up to 100-fold and the decrease was inversely proportional to the effective molecular size. The in vitro activities such as ribosome-inactivating activity and cytotoxicity were also decreased. However, the in vivo abortifacient activity was, slightly decreased, unchanged, or even enhanced in some preparations. PEG5k modification had little effect on immunogenicity. However, PEG20k modification significantly reduced immunogenicity. All PEG20k modified TCS mutants induced lower level IgG and IgE antibodies. In particular, PEG20k-KQ and PEG20k-K173C induced weaker systemic anaphylaxis reaction in guinea pigs. In conclusion, the present results suggest that PEG20k is better than PEG5k for reducing immunogenicity and prolonging plasma half-life. The conjugate can become a better therapeutic agent.  相似文献   

13.
Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG20k-S7C, PEG20k-K173C, and PEG20k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life.  相似文献   

14.
PEGylation of antibodies is known to increase their half-life in systemic circulation, but nothing is known regarding whether PEGylation can improve the inhibitory potency of antibodies against target receptors. In this paper, we have examined this question using antibodies directed to Sialoadhesin (Sn), a macrophage-restricted adhesion molecule that mediates sialic acid dependent binding to different cells. Anti-Sn monoclonal antibodies (mAbs), SER-4 and 3D6, were conjugated to PEG 5 kDa or and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. Following purification of PEGylated mAbs by anion exchange chromatography, it was shown that PEGylation had little or no effect on antigen binding activity but led to a dramatic increase in inhibitory potency that was proportional to both the size of the PEG and the degree of derivatization. Thus, PEGylation of antibodies directed to cell surface receptors could be a powerful approach to improve the therapeutic efficacy of antibodies, not only by increasing their half-life in vivo, but also by increasing their inhibitory potency for blocking receptor-ligand interactions.  相似文献   

15.
The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.  相似文献   

16.
利用人白细胞介素11(hIL-11)无半胱氨酸(Cys)残基这一特点,通过定点突变将一个Cys残基引入hIL-11的N末端。然后,利用与Cys 巯基特异性反应的mPEG-马来酰亚胺将mPEG偶联到预先选定的位点,经层析纯化得到hIL-11的定点PEG修饰物。利用依赖型细胞株7TD1测定其生物学活性,结果表明,其体外生物学活性保持原有hIL-11活性的30%左右。定点聚乙二醇修饰方法为定向改造hIL-11,提高其药效的应用研究打下基础。  相似文献   

17.
目的:设计构建集成干扰素突变体IIFN72C并进行聚乙二醇定点修饰,以获得高活性的长效干扰素分子。方法:利用蛋白质分子同源模建,选择在集成干扰素分子IIFN的第72位引入半胱氨酸残基构成集成干扰素突变体IIFN72C。诱导表达后经包涵体变复性和层析纯化,与单甲氧基聚乙二醇(mPEGMAL)定点偶联。修饰产物经纯化后,以SDSPAGE考察其纯度,用WISHVSV系统进行生物活性测定。结果:IIFN72C以包涵体形式表达,表达量占菌体总蛋白的30%以上,比活性与突变前相当;修饰产物大多数为单修饰体,纯化后纯度大于98%,比活性保留约为修饰前的8%。结论:成功设计并表达IIFN72C用于PEG定点修饰,修饰产物活性保留得以提高。  相似文献   

18.
聚乙二醇(PEG)定点修饰蛋白药物是针对蛋白特定基团特定位点的修饰,相比于非定点随机修饰的特点是PEG修饰位点的单一与确定,避免了修饰异构体的干扰,能较好的保留药物体内外活性;修饰产物组成均一、性质稳定,便于质量控制,降低由修饰异构体引起的潜在的安全性风险,并很大程度上提高得率,降低成本。已有PEG定点修饰蛋白药物上市,还有部分处于临床试验阶段。本文综述了PEG定点修饰蛋白药物的技术研究与临床进展,包括PEG定点修饰剂、定点修饰方法、PEG定点修饰的上市和临床药物及面临的问题,并展望了PEG修饰技术未来的发展前景。  相似文献   

19.
PEGylation can improve the therapeutic efficacy of proteins by increasing serum half-life of proteins and reducing immunogenicity and antigenicity. However, PEGylation results in a substantial loss of the bioactivity of proteins due to the steric hindrance of polyethylene glycol (PEG). Dimerization of the proteins is an efficient approach to increase the bioactivity of the PEG-protein conjugates. Here, staphylokinase (SAK) was used due to its therapeutic potential for coronary thrombolysis. SAK dimers (dSAK) were prepared by engineering cysteine residue at the C-terminus of SAK and dimerization of the cysteine residue with 1,4-bismaleimidobutane. PEG aldehyde was used for site-specific PEGylation of dSAK at one of its two N-termini. Structural analysis indicated that dimerization of SAK can decrease the steric hindrance of PEG and increase the binding affinity of PEG-SAK to plasminogen. Dimerization of SAK increased the relative bioactivity of PEG-SAK from 39.0% to 62.0%. Therefore, site-specifically PEGylated dSAK at one of its two N-termini has higher bioactivity than the N-terminal PEGylated SAK.  相似文献   

20.
Peptides as therapeutic substances are efficient agents in the treatment of several diseases. However, they often have to be chemically modified in order to be suited as therapeutic agent. Conjugation to large carrier molecules is often required. A critical step is the identification of available sites for chemical reaction, without influencing bioactivity. Peptide 238/S1 with the sequence NH(2)-PYWKWQYKYD-COOH previously selected from a combinatorial decapeptide library, has the ability to block inhibitory antibodies against blood clotting factor VIII (FVIII) and therefore, it constitutes a lead for developing a drug to treat patients suffering from development of such antibodies. The aims of this study were (i) to identify sites of the peptide, which are suited for modification without losing bioactivity and (ii) to find out the influence of molecular size of polyethylene glycol (PEG) for bioactivity of the peptide. The contribution of each amino acid residue to biological functionality was investigated by mutational analysis. This method confirmed that the N-terminus is crucial for activity, whereas both lysine residues could be exchanged by other L-amino acids. Using mutational analysis it was possible to identify peptides with higher reactivity compared to the wild type 238/S1. PEGylation experiments demonstrated that conjugation of the peptide to PEG 20,000 resulted in a loss of reactivity, while PEG 5,000 could maintain the bioactivity when conjugated in a site directed manner. The peptide lost its neutralization properties when PEG was coupled to the N-terminus, again indicating that this part of the peptide is important for functionality.  相似文献   

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