蛇毒蛋白Echistatin的C端突变基因的构建,表达及其活性研究

通过ＰＣＲ定点突变的技术,将蛇毒蛋白Ｅｃｈｉｓｔａｔｉｎ基因的Ｃ端进行了突变（Ａｌａ４８→Ａｒｇ４８→,Ｔｈｒ４９→Ｖａｌ４９）,模拟纤维蛋白Ｎ端的四肽（Ｇｌｙ－Ｐｒｏ－Ａｒｇ－Ｖａｌ）,以期增加Ｅｃｓ（Ｅｃｈｉｓｔａｔｉｎ）的活性,突变的基因重组到表达质粒ｐＪＣ２６４上,经ＩＰＴＧ诱导,以ＣｈｅＹ－Ｅｃｓ融合蛋白方式进行了表达,表达量占菌体总蛋白的１５￣２０％,Ｓｅｐｈａｄｅｘ Ｇ－７５初步纯化     

p16抑癌基因定点突变及其在大肠杆菌中的表达与纯化

为了研究错义突变对ｐ１６功能的影响,应用ＰＣＲ体外定点突变方法对ｐ１６ｃＤＮＡ进行体外定点突变,并将野生型和突变型ｐ１６ｃＤＮＡ克隆于ｐＧＥＸ－５Ｔ载体,在大肠杆菌中经ＩＰＴＧ诱导表达,Ｗｅｓｔｅｒｎ印迹鉴定确证表达．而后用谷胱甘肽－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化野生型和突变型ｐ１６融合蛋白．得到了第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）突变的ｐ１６－Ｐ４８突变体,并在大肠杆菌中表达了４２ｋＤ的ＧＳＴ－ｐ１６和ＧＳＴ－ｐ１６Ｐ４８Ｌ融合蛋白．最后经纯化得到了野生和Ｐ４８Ｌ突变的ｐ１６融合蛋白     

天花粉蛋白Y14F/R22L定点突变及其活性研究

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．     

青霉素G酰化酶基因Ser177的定点突变

本研究用缺口以链法对肠杆菌青霉素Ｇ酰化酶（ＰＧＡ）基因Ｓｅｒ１７７进行寡核苷酸定点突变。通过ＮＩＰＡＢ（２－硝基－５－苯乙酰胺苯甲酸）试纸法筛选和测序鉴定,获得突变体Ｃｙｓ１７７,Ｇｌｙ１７７,Ａｒｇ１７７和Ａｓｎ１７７。它们的ＰＧＡ活性均已丧失。酶蛋白电泳分析表明突变体蛋白在体内正常表达。推测ＰＧＡ Ｓｅｒ１７７秀可能位于酶底物结合中心,是酶活性所必需,不能被置换。     

蛇毒锯鳞蝰素基因Leul4－Lys15－Glu16的定点突变

本研究的目的是利用蛋白质工程定点突变的方法,在蛇毒锯鳞蝰素基因分子上增加另一个保守序列ＲＧＤ（１４位精氨酸残基,１５位甘氨酸残基,１６位天冬氨酸残基）,以其增加该分子的生物活性,并探讨蛋白质一级结构,空间结构和功能的关系。在质粒ｐＪＣ２６４的基础上,利用ＰＣＲ定点突变方法,对蛇毒锯鳞蝰素基因Ｌｅｕ１４－Ｌｙｓ１５－Ｇｌｕ１６进行定点突变,使相应的ＤＮＡ片段变成表达Ａｒｇ１４－Ｇｌｙ１５－Ａｓｐ１６的核苷酸顺序,经酶切和ＤＮＡ测序鉴定正确。ＣＮＢｒ裂解后,用反相ＨＰＬＣ分析,分离制备突变体蛇毒锯鳞蝰素,制备的突变体蛇毒锯鳞蝰素的Ｎ－末端１０个氨基酸残基与天然蛇毒锯鳞蝰素的相同。在人的富含血小板血浆测活体系中,经１０μｍｏｌ／Ｌ的ＡＤＰ诱导,突变体蛇毒锯鳞蝰素的ＩＣ５０为３．０×１０~（－７）ｍｏｌ／Ｌ;重组野生型蛇毒锯鳞蝰素的ＩＣ５０为４．０×１０~（－７）ｍｏｌ／Ｌ;天然蛇毒锯鳞蝰素的ＩＣ_（５０）为２．８×１０~（－７）ｍｏｌ／Ｌ。此外,就蛇毒锯鳞蝰素中保守序列Ａｒｇ－Ｇｌｙ－Ａｓｐ以及其空间构象对蛇毒锯鳞蝰素抑制血小板凝集之间的关系进行了讨论。     

G蛋白Rap1活化新机制：GEFs研究进展

小分子Ｇ蛋白Ｒａｓ超家庭成员都是通过与鸟苷酸交换因子（ｇｕａｎｉｎｅ ｎｕｃｌｅｏｔｉｄｅ ｅｘｃｈａｎｇｅ ｆａｃｔｏｒｓ,ＧＥＦｓ）结合而被活化,ＧＥＦｓ能增加这类小分子Ｇ蛋白对ＧＴＰ的摄取,并使之形成有活性的ＧＴＰ结合构象。Ｒａｐ１是Ｒａｓ样的小分子Ｇ蛋白。迄今为止,已发现Ｇ３Ｇ、ＣａｌＤＡＧ－ＧＥＦ１、Ｅｐａｃ、ｃＡＭ－ＧＥＦ１、ｃＡＭＰ－ＧＥＦⅡ、ｎＲａｐＧＥＰ、ＧＦＲ可特异的活化Ｒａｐ     

蟾蜍细胞溶素的纯化及其生化特性研究

利用阴离子交换和凝胶过滤柱层析等方法对蟾蜍卵黄外被细胞溶素进行了分离纯化,获得了高纯度的样品．该酶的质量为３２ｋＤ,其特异性ＭＣＡ－人工合成底物为Ｂｏｃ－Ｇｌｎ－Ａｒｇ－Ａｒｇ－ＭＣＡ,能被ＤＦＰ、ＳＢＴＩ、ｌｅｕｐｅｐｔｉｎ和ｐ－ＡＭＰＳＦ等蛋白酶抑制剂所强烈抑制,但不受ｃｈｙｍｏｓｔａｔｉｎ、ｂｅｓｔａｔｉｎ、Ｅ－６４和ＥＤＴＡ等的影响,表明该酶是一种丝氨酸类型的蛋白酶     

用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度

用寡核苷酸诱导的定点突变方法构建了葡萄糖异构酶基因的突变体（Ｎ１８４Ｄ和Ａ１９８Ｃ）。含突变体的重组质粒ｐＴＫＤ－ＧＩ１（Ｎ１８４Ｄ）和ｐＴＫＤ－ＧＩ２（Ａ１９８ｃ）在Ｅ．ｃｏｌｉＫ３８菌株中表达,用ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦＦ和Ｓｅｐｈａｃｒｙｌ Ｓ－３００ＨＲ柱层析分离纯化突变酶。与野生型葡萄糖异构酶比较实验表明：（１）突变酶Ｎ１８４Ｄ的最适ｐＨ值下降了１个单位;等电点下降了０．６个单位     

白细胞介素—2分子α螺旋B中的活性相关区域

根据人白细胞介素－２（ＩＬ－２）α螺旋Ｂ中氨基酸残基的空间分布选择性地突变了一些氨基酸残基,结果发现：５７Ｇｌｎ→Ｇｌｕ,６２Ｇｌｕ→Ｌｅｕ,６２Ｇｌｕ→Ａｒｇ和６５Ｐｒｏ→Ａｒｇ这些替换均使ＩＬ－２活性显著降低或丧失,而６３Ｌｅｕ→Ｓｅｒ或６４Ｌｙｓ→Ａｌａ对ＩＬ－２活性影响不大。从受体竞争抑制结合实验结果可知,上述不表现活性的突变体也同时丧夫了与高亲和力受体的结合能力,这说明α螺旋Ｂ中这些位点     

贵州省西北部“百里杜鹃林”的土壤生态条件

贵州省西北部“百里杜鹃林”的土壤生态条件庞纯焘,宋铭荷,田光普（贵州教育学院,贵阳５５０００３）（贵州农学院,贵阳５５０００６）ＳｏｉｌＥｃｏｌｏｇｉｃａｌＣｏｎｄｉｔｉｏｎｓｉｎｔｈｅ＂ＨｕｎｄｒｅｄＬｉ（５００ｍ）ＡｚａｌｅａＦｏｒｃｓｔ＂ｉｎＮｏｒｔｈｗｅｓｔＧｕｉｚｈｏｕＰｒｏｖｉｎｃｅ￥ＰａｎｇＣｈｕｎｔａｏ;ＳｏｎｇＭｉｎｇｈｅ（ＧｕｉｚｈｏｕＥｄｕｃａｔｉｏｎＣｏｌｌｅｇｅ,Ｇｕｉｙａｎｇ５５０００３）,ＴｉａｎＧｕａｎｇｐｕ（ＧｕｉｚｈｏｕＡｇｒｉｃｕｌｔｕｒａｌＣｏｌｌｅｇｅ,Ｇｕｉｙａｎｇ５５０００６）．ＣｈｉｎｅｓｅＪｏｕｒｎａｌｏｆＥｃｏｌｏｇｙ,１９９３,１２（１）：４９－５２。Ｔｈｅｒｅｉｓａｖａｓｔａｒｅａｏｆａｚａｌｅａ（Ｒｈｏｄｏｄｅｎｄｒｏｎｓｐｐ．）ｆｏｒｅｓｔｉｎＮｏｒｔｈｗｅｓｔＧｕｉｚｈｏｕＰｒｏｖｉｎｃｅ,Ａｔｆｌｏｗｅｒｉｎｇｓｅａｓｏｎｉｎｓｕｍｍｅｒ,ｉｔｂｅｃｏｍｅｓａｒｅｇｉｏｎｏｆｔｏｕｒｉｓｔａｔｔｒａｃｔｉｏｎ．Ｔｈｉｓｒｅｇｉｏｎｉｓｓｏｆａｓｃｉｎａｔｉｎｇｔｈａｔｐｅｏｐｌｅｃａｌｌｉｔ＂ＨｕｎｄｒｅｄＬｉ（５００ｍ）ＡｚａｌｅａＦｏｒｅｓ     

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo

The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.     

High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.     

Targeting of the mammalian nucleoporin p62 to the nuclear envelope in the yeast Saccharomyces cerevisiae and HeLa cells.

We have analyzed the sorting of the mammalian nucleoporin p62 in human culture cells and in the yeast Saccharomyces cerevisiae. To this end, gene fusions were generated that carry Aequorea victoria green fluorescence protein and defined portions of p62. Upon transient gene expression fluorescent fusion proteins were localized in HeLa cells. Likewise, fusion proteins were studied in S. cerevisiae using wild-type as well as mutant cells that cluster nuclear pore complexes. Our results demonstrate that evolutionarily distant organisms, such as humans and yeasts, recognize the same sequence elements of p62 for sorting to the nuclear envelope. Specifically, the entire sequence of p62 or its complete C-terminal domain targeted fusion proteins to the nuclear membranes. In contrast, truncations of the C-terminal domain or the N-terminal segment of p62 failed to associate with the nuclear envelope in either organism. In HeLa cells overexpression of several p62-containing fusion proteins resulted in nuclear fragmentation. The C-terminal domain of p62 caused this effect, and amino acid residues 477 to 525 were sufficient to induce aberrant nuclei. Thus, overexpression of 49 amino acid residues located at the C-terminal tail of p62 interferes with the nuclear integrity in human culture cells.     

三叶因子Bm-TFF2突变体在原核体系中的表达及促细胞迁移活性

大蹼铃蟾三叶因子Bm-TFF2具有较人TFF2更强的促细胞迁移和抗凋亡活性。该研究利用RT-PCR方法扩增得到野生型Bm-TFF2的基因,然后分别构建N端、C端和分子中两个精氨酸突变的突变体,最后连接表达载体产生pET32a(+)/Bm-TFF2突变型重组质粒,转入大肠杆菌中,经37℃培养,IPTG诱导,其融合蛋白主要存在于包涵体中,用组氨酸标签的亲合柱纯化溶解后的包涵体上清,进一步用RP-HPLC纯化得到硫氧还蛋白(TRX)/Bm-TFF2突变型融合蛋白。通过SDS-PAGE和Westernblotting检测分析其纯度和特异性。最终,从1L培养基中得到20mg纯度为95%的三种重组突变型融合蛋白。三种突变型重组蛋白都具有剂量依赖性的促细胞迁移活性,并且其活性无显著差异。该研究为进一步研究Bm-TFF2结构和功能的关系以及揭示其作用的分子机制奠定了基础。     

杜氏盐藻核基质结合区结合蛋白的细胞定位

为研究核基质结合区结合蛋白的功能及调控机制,PCR扩增杜氏盐藻MBP的cDNA全长序列及N端和C端序列,与绿色荧光蛋白基因融合构建真核表达载体,脂质体转染CHO细胞,Western blotting和荧光显微镜检测基因表达情况和细胞定位。结果显示:MBP及N端和C端融合蛋白成功在CHO细胞表达,MBP和C端部分定位于细胞核且聚集于核仁,N端部分分布整个细胞,说明MBP定位于细胞核且细胞定位信号位于C端,MBP可能与rRNA前体结合发挥作用。     

重组人睫状神经营养因子突变体的构建、表达及生物活性分析

目的:通过对原始CNTF的突变改造,降低表达蛋白的免疫原性,同时增加其稳定性,以期能用于临床治疗肥胖症。方法:以原始CNTF为模板,去掉N端的12个氨基酸、C端的14个氨基酸,并将C端末2个氨基酸点突变为极性较强的赖氨酸,在大肠杆菌中表达,获得CNTF-T突变体。结果:基因序列分析与原设计吻合,目的蛋白表达量占全菌体的35%左右,表达蛋白以包涵体形式存在,经变性、复性、DEAE-FF纯化,纯度可达95%以上,小鼠体内生物活性测定,能明显抑制小鼠体重增长,且CNTF-T的生物活性比原始序列CNTF的活性高。结论:突变的CNTF-T有望成为新一代的生物减肥制剂应用于临床。     

Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.     

Improved solubility of TEV protease by directed evolution

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.