大肠杆菌AroG蛋白F209S突变体对苯丙氨酸反馈抑制作用的影响(英文)

在大肠杆菌中 ,80 %的 3 脱氧 D 阿拉伯 庚酮 7 磷酸 (3 deoxy D arabino heptulosonate 7 phosphate,DAHP)合酶由aroG基因编码。分别以大肠杆菌K1 2及其抗苯丙氨酸类似物的突变体总DNA为模板 ,以PCR方法扩增得到aroG基因及其突变体。基因测序结果表明抗苯丙氨酸类似物的突变体 ,其aroG基因核苷酸 62 5位发生了T→C的点突变 ,从而使AroG蛋白的 2 0 9位氨基酸由Ser取代了Phe。aroG基因及其突变体克隆到表达质粒 pTrc99A上 ,在大肠杆菌JM 1 0 5中进行表达 ,表达产物的SDS PAGE上可以看到分子量相当明显的条带 ;菌体粗提物中DAHP合酶的活性提高了 1 .8倍 ;酶活抗性检测表明AroG蛋白突变体在一定程度上解除了苯丙氨酸的反馈抑制作用 ;与含K1 2aroG基因的JM1 0 5细胞相比 ,含aroG基因突变体的JM1 0 5细胞可以在含高浓度苯丙氨酸类似物的培养基上生长。     

大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析

为了获得具有抗反馈抑制性质的大肠杆菌磷酸甘油酸脱氢酶（PGDH, d-3-phosphoglycerate dehydrogenase, EC 1.1.1.95）,通过对其碱基序列和蛋白质结构分析,用PCR突变法构建突变酶M1（缺失第410位氨基酸）、M2（缺失407~410位氨基酸）、M3（缺失337~410位氨基酸）。M0(野生型)及各突变型基因与pET22b(+)载体连接后,表达融合蛋白。在非变性条件下,由NTA-Ni镍离子螯合亲和层析柱纯化野生型和突变体的酶蛋白。酶活性测定结果表明,M1、M2蛋白酶均保持了原有的野生型磷酸甘油酸脱氢酶活性,且部分解除了终产物L-丝氨酸的反馈抑制作用;M3蛋白酶完全解除了终产物的反馈抑制作用,但酶本身的催化活性略有降低（为野生型的83%）。M0、M1、M2菌株PGDH与L-丝氨酸结合的Ki值分别约为7 μmol/L、20 μmol/L、50 μmol/L,说明该酶C-末端1~4个氨基酸残基对L-丝氨酸和调控区的结合有重要影响。     

重组链激酶及其三种突变体的活性研究

利用PCR技术从Streptococucspyogenes的基因组DNA中扩增了链激酶的编码基因ska,并进行了序列分析 ,利用基因删除及定点位变技术获得了删除了C-末端 42个氨基酸残基编码区的突变链激酶基因skaΔC42 ,第 5 9位Lys残基突变为Glu的突变链激酶基因skaK5 9E以及删除C-末端 42个氨基酸且第 5 9位Lys残基突变为Glu的突变链激酶基因skaΔC42K5 9E ,将ska及其三种突变体分别克隆到表达载体pET 1 5b上 ,构建分别表达野生型链激酶 (SK)、C-末端缺失 42个氨基酸残基的突变体 (SKΔC42 )、第 5 9位Lys残基突变为Glu的突变体 (SKK5 9E)及C-末端缺失 42个氨基酸且第 5 9位Lys残基突变为Glu突变体 (SKΔC42K5 9E)的表达载体pSK ,pSKΔC42 ,pSK K5 9E ,pSKΔC42K5 9E ,分别转化E .coliBL2 1 (DE3) ,IPTG诱导后在大肠杆菌中实现了高效表达 ,经亲和层析、离子交换层析及分子筛层析 ,获得了rSK、rSKΔC42、rSKK5 9E及rSKΔC42K5 9E ,活性分析表明rSK与其三种突变体具有相同的比活性。     

人凋亡相关基因TFAR19缺失突变体的原核表达、产物纯化及鉴定

构建TF 1细胞凋亡相关基因 19(TF 1cellapoptosisrelatedgene 19,TFAR19)缺失突变体的原核表达载体 ,获取缺失突变体蛋白 ,用于TFAR19促凋亡分子机理的研究 .从真核表达载体pcDI TFAR19扩增出野生型TFAR19和 4个缺失突变体 ,重组到原核表达载体pGEX 4T 2 .经亲和层析方法对缺失体蛋白进行纯化后 ,再利用凝胶过滤的方法进一步纯化 .利用抗GST和抗TFAR19的单克隆抗体对蛋白进行免疫学鉴定 .用白血病细胞株HL 6 0检测蛋白活性 .成功地克隆并重组了野生型TFAR19及缺失突变体 pGEX 4T 2表达载体 ,对融合蛋白的表达条件进行了优化 .SDS PAGE结果显示 ,各个缺失突变体融合蛋白均有较高水平的表达 .免疫学检测证实获得了正确的表达产物 .活性检测证实 ,野生型TFAR19和缺失突变体 4可以明显促进去血清诱导的HL 6 0细胞凋亡 ,第 6外显子可能是一个与TFAR19促凋亡活性密切相关的功能结构域     

低毒且高效杀伤人肿瘤细胞的新型人肿瘤坏死因子突变体的构建

采用定点突变的方法构建了两株新型人肿瘤坏死因子突变体MT1 (32Trp157Phe)和MT2(2Lys30Ser32Trp157Phe), 并在大肠杆菌中得以高效表达, 表达量占菌体总蛋白的50%且为可溶性蛋白. 表达产物经蛋白印渍法检测, 均能与抗野生型hTNF-a单抗结合且经分离纯化后样品的纯度在95%以上. 各突变体虽然对鼠成纤维瘤细胞L929的生物学活性比野生型下降了4～5个数量级, 但却能高效杀伤人肿瘤细胞, 杀伤程度与野生型相差不明显. 测定突变体MT1的LD50值降至野生型的0.34%, 而MT2对小鼠30%致死剂量则低于野生型LD50的1/700.     

低毒且高效杀伤人肿瘤细胞的新型人肿瘤

采用定点突变的方法构建了两株新型人肿瘤坏死因子突变体MT1 (32Trp157Phe)和MT2(2Lys30Ser32Trp157Phe), 并在大肠杆菌中得以高效表达, 表达量占菌体总蛋白的50%且为可溶性蛋白. 表达产物经蛋白印渍法检测, 均能与抗野生型hTNF-a单抗结合且经分离纯化后样品的纯度在95%以上. 各突变体虽然对鼠成纤维瘤细胞L929的生物学活性比野生型下降了4~5个数量级, 但却能高效杀伤人肿瘤细胞, 杀伤程度与野生型相差不明显. 测定突变体MT1的LD50值降至野生型的0.34%, 而MT2对小鼠30%致死剂量则低于野生型LD50的1/700.     

重组刺桐胰蛋白酶抑制剂a在大肠杆菌中的表达和纯化

为了大量制备重组刺桐胰蛋白酶抑制剂a(rETIa) ,对构建的基因工程菌株E .coliBL2 1(DE3)pET2 2b mETIa进行了表达条件的优化 .用摇瓶培养 ,rETIa蛋白占菌体总蛋白 4 0 %以上 .经破碎菌体 洗涤包涵体 溶解包涵体 复性初步纯化后 ,再经二步柱层析纯化获得电泳纯的rETIa蛋白 .测定了rETIa对胰蛋白酶、胰凝乳蛋白酶、组织型纤溶酶原激活因子缺失突变体 (NTA)的抑制活性 .     

灰葡萄孢菌及其抗短梗霉素突变体AUR1基因序列及其酶活性的分析

【目的】探讨灰葡萄孢菌及其抗Ab A突变体AUR1基因序列与IPC合成酶活性的关系。【方法】通过分子生物学方法测定野生型及突变体的AUR1的基因序列,高效液相荧光色谱法测定IPC合成酶活力,苯甲酰化法测定神经酰胺含量。【结果】AUR1基因序列和IPC合成酶活性测定表明4株不同的突变体均产生了对IPC合成酶抑制剂Ab A的抗性,它们的突变类型为:(1)AUR1序列中缺失内含子;(2)AUR1序列中缺失内含子和P155S氨基酸突变;(3)AUR1序列中缺失内含子和V33A的氨基酸突变;(4)AUR1序列中缺失内含子和P155S、S177P、F237L的氨基酸突变。AUR1缺失内含子和既缺失内含子又伴随P155S氨基酸突变的突变体的Ab A抗性较强。神经酰胺含量测定表明野生型IPC合成酶被抑制,导致神经酰胺积累,而突变体则能抵抗Ab A对IPC合成酶的抑制作用。【结论】AUR1基因中的内含子对IPC合成酶的调控起重要的作用。Ab A通过抑制IPC合成酶引起神经酰胺积累,IPC合成酶是鞘脂代谢的关键酶。     

提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用

将B. circulans 251β-CGTase应用于海藻糖制备,海藻糖转化率从50. 4%提高至71. 9%。为进一步提高底物的转化率,运用易错PCR-高通量筛选技术筛选对以麦芽糖为歧化反应受体的亲和性提高的B. circulans 251β-CGTase突变体。利用低底物浓度的96孔板4,6-亚乙基-对硝基苯-α-D-麦芽七糖苷(EPS)显色法,最终筛选得到了一株对麦芽糖亲和性提高的突变体M234I。将野生型β-CGTase和突变体酶M234I进行蛋白质纯化,测定其酶学性质。结果表明,突变体的比活为345. 25U/mg,野生型则为357. 63U/mg;突变体M234I对麦芽糖的K_m为0. 258 2mmol/L,仅为野生型(0. 474 9mmol/L)的54. 4%,对麦芽糖的亲和性显著提高;突变体的最适温度、最适p H较野生型未发生较大变化。以麦芽糊精(DE值16)为底物,将突变体M234I用于多酶复配体系生产海藻糖,酶反应结果表明海藻糖的转化率最高达74. 9%,较野生型β-CGTase提高约3%。     

葡萄球菌肠毒素 B 突变体的构建及其抗肿瘤活性分析

目的：通过对葡萄球菌肠毒素B（SEB）32位His进行定点突变,获得抑瘤效果显著增强的SEB突变体。方法：利用基因定点突变的方法,将SEB的32位His替换为Asn,将重组质粒转入大肠杆菌中诱导表达,用CM弱阳离子层析柱纯化获得重组蛋白,用SDS-PAGE和Western印迹对其进行鉴定,并用增殖实验检测其丝裂原活性,通过MTS法检测其体外抗肿瘤活性。结果：构建并高效表达了突变体蛋白SEB（H32N）,纯化获得了足够纯度的突变体蛋白;体外实验表明,在相同浓度下,SEB（H32N）的丝裂原活性及体外抗肿瘤活性明显优于野生型SEB。结论：同野生型SEB相比,突变体蛋白SEB（H32N）对肿瘤生长的抑制作用得到了提高。     

C-terminal region of the active domain enhances enzymatic activity in dinoflagellate luciferase.

The dinoflagellate luciferase of Lingulodinium polyedrum has three catalytic domains in its single polypeptide chain (M(r) = 137 kDa), and each 42 kDa domain is enzymatically active. Deletion mutants for N- or C-terminal regions of domain 3 of the luciferase, ranging from 29 to 38 kDa, were constructed and expressed in E. coli cells. The activities of N-terminal deleted mutants were above 20% of wild type, but showed different pH-activity profiles. By contrast, the activities of C-terminal deleted mutants decreased drastically to below 1% of wild type, although their pH-activity profiles and spectra were identical to those of wild type L. polyedrum luciferase. These results indicate that the C-terminal region of this enzyme could be important for the bioluminescence reaction, although based on crystal structure of the luciferase domain, this region does not contain active or regulatory sites.     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.     

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.     

大肠杆菌苯丙氨酸合成途径关键酶基因pheA的克隆与表达

为了通过基因工程手段来增加苯丙氨酸的生物产量,利用ＰＣＲ方法从大肠杆菌中克隆了抗反馈抑制突变型及野生型的ｐｈｅＡ基因,进行了核苷酸序列分析,并利用高效的原核表达载体ＰＢＶ２２０对ｐｈｅＡ基因编码的突变型及野生型分支酸变位酶／预苯酸脱水酶（ＣＭ／ＰＤ）进行了表达。序列分析表明突变型基因碱基第５８０位由Ｔ变为Ｃ,相应氨基酸由Ｖａｌ变为Ａｌａ,ＳＤＳ－ＰＡＧＥ图谱扫描分析表明目的蛋白ＣＭ／ＰＤ的表达量占全菌体蛋白的４３％,占上清总蛋白的５７％。酶活性测定表明其ＣＭ和 ＰＤ活性分别提高了 １５．５和６．７倍,产酸量也有了一定的提高,为构建产苯丙氨酸的生物工程菌奠定了基础。     

The structural interpretations of residue Ser297 in catalytic efficiency of Escherichia coli phenylalanine aminotransferase

Escherichia coli phenylalanine aminotransferase (ecPheAT) catalyzes the biosynthesis of phenylalanine and tyrosine. The crystal structure of ecPheAT was determined in our previous study. The comparison of the 3-D structure of several aminotransferases revealed that the residue at position 297 plays an important role in enzyme function. Analysis of activities and kinetic parameters of wild type and mutant ecPheATs suggested that the residue Ser(297) was structurally selected for better catalytic efficiency. Computational modeling of ecPheAT mutants further suggested that Ser in position 297 could make ecPheAT easy with change of conformation from open form to closed form.     

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein.

The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.     

Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.     

The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25°C in 50mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K(i) values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 μM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K(i) values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.