天冬氨酸酶E.coli融合表达研究

以大肠杆菌基因组DNA为模板,设计引物扩增得到天冬氨酸酶基因,将其重组于胞内融合表达型T载体中,重组质粒转化表达宿主大肠杆菌BL21(DE3)。SDS-PAGE分析表明,工程菌经IPTG诱导,表达大量表观分子量约75kD的融合蛋白。经试验,工程菌细胞具有较高的天冬氨酸酶活性,融合形式的酶最适温度37℃,最适pH8.5,融合伴侣DsbA的存在对酶活没有影响。     

AspC基因导入前后E.coli BL21蛋白质组的解析

为了考察表达天冬氨酸转氨酶工程菌在转基因前后蛋白质水平的差异变化,采用固相pH梯度-SDS聚丙烯酰胺双向凝胶电泳对转基因前后的大肠杆菌（E．coli BL21）的总蛋白进行分离,银染、显色后,使用2D蛋白质图象分析系统Image Master 2D Platinum 5．0和SWISS-2D PAGE蛋白质组数据库对双向电泳图谱进行分析,识别了近600个蛋白点,比较分析了与苯丙氨酸合成途径相关的关键蛋白的差异,初步探讨了AspC基因的导入后大肠杆菌蛋白质水平的精细调控。     

E.coli Rho因子的结构与功能

Ｅ．ｃｏｌｉＲｈｏ因子的结构与功能赖兵张海（北京大学生命科学学院生物化学与分子生物学系,北京１００８７１）关键词Ｒｈｏ因子ＡＴＰａｓｅ转录过程中起决定作用的序列,蛋白质因子及它们之间的相互作用,一直是生物化学家和分子生物学家关注的焦点之一。由于真核生...     

APC基因结构及功能异常与人类肿瘤的关系

腺瘤样结肠息肉病易感基因是１９９１年从结肠癌中克隆到的一个新的抑癌基因,定位于５号染色体长臂。ＡＰＣ基因产物功能不茂清楚,发现其可调节ｃ－ｍｙｃ基因ｍＲＮＡ的成熟和ｒａｓ基因的表达,与蛋白质相互作用有关。     

荧光假单胞菌天冬氨酸转氨酶的基因克隆及其在大肠杆菌中的表达

采用克隆基因测序技术,从荧光假单胞菌GcM5-1A基因组文库中筛选到了天冬氨酸转氨酶的编码基因aspC。通过聚合酶链式反应（PCR）扩增目的基因,插入pET-15b构建重组表达质粒pET-15bAAT,转化E.coli BL21(DE3),IPTG诱导天冬氨酸转氨酶在大肠杆菌中高效表达,利用亲和层析法初步分离纯化了重组蛋白。生物活性分析表明,纯化的重组天门冬氨酸转氨酶具有氨基转移活性。     

不同猪种E.coli F18受体基因的多态性

采用PCR—RFLP技术检测了大约克、长白、杜洛克、宁乡、沙子岭和大围子6个品种共867头猪的E.coli F18受体(ECF18R)基因座的遗传变异。结果表明：Hin6 Ⅰ-RFLP位点上,大约克、长白、杜洛克3个外来猪种均存在多态,且以敏感型(GG型和AG型)居多,平均占94％,3个外来猪种的G等位基因频率平均为0．76,AA抗性型个体占少数,平均为6％,猪群中M307处G→A的突变频率并不高。宁乡、沙子岭和大围子3个本地猪种的所有检测样品都表现为GG型,在该位点上均不存在G→A的突变。各猪种ECF18受体基因座的PCR—RFLP基因型分布X^2检验结果表明,每个外来猪种ECF18受体基因座的PCR—RFLP基因型分布与3个本地猪种的相比均差异显著或极显著,3个本地猪种间的ECF18受体基因座的PCR—RFLP基因型分布完全一致。外来猪种间只有长白与杜洛克各基因型的分布差异显著,其余均不显著。     

NGAL的结构与功能研究进展

NGAL（neutrophil gelatinase-associated lipocalin)是一种新的lipocalin,其结构与功能的研究进展为人瞩目,目前认为NGAL具有运输疏水性分子,调节明胶酶B（gelatinaseB或matrix metalloproteinase-9,MMP-9)活性等功能,并可能参与免疫炎症反应以及肿瘤的发生发展,特别是肿瘤的浸润转移等过程,最近,NGAL蛋白的空间结构已被研究清楚,这对于促进NGAL生物学作用或功能的研究。特别是对其新的生物学作用或功能的探索,比如与肿瘤的关系（NGAL可能是一种新的癌基因或促癌基因）将具有十分重要的意义。对此作者进行了综合评述。     

E.coli核糖体蛋白质S12依赖链霉素突变抑制λN基因表达的分子机制

本文利用ＰＣＲ方法克隆了Ｅ．ｃｏｌｉＴ８３依赖链霉素（ｓｔｅｒｐｔｏｍｙｅｉｎｄｅｐｅｎｄｅｎｔ,Ｓｍ＾ｄ）菌株中编码突变的核糖体蛋白质Ｓ１２的ｒｐｓＬ＾ｄ基因,并进行了ＤＮＡ序列分析,发现第４２位密码子由编码赖氨酸（Ｌｙｓ,Ｋ）的ＡＡＡ变为编码谷氨酰胺（Ｇｌｎ,Ｑ）的ＣＡＡ。依据Ｇａｒｎｉｅｒ原理预测了突变对Ｓ１２蛋白质二级结构形成趋势的影响,结果表明,突变使第４２位氨基酸及其相连肽段的β－转折     

杆状病毒几丁质酶基因结构与功能的研究进展

杆状病毒几丁质酶基因是杆状病毒的非必需基因,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶,该酶N端具信号肽,中部是酶的活性区,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的,同时它还是原组织蛋白酶(pro-V-Cath)的分子伴侣,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

MyoG基因结构与功能研究进展

肌细胞生成素(myogenin,MyoG)是生肌调节因子MRFs(MyoD、MyoG、Myf5和MRF4)家族的一员,它的表达具有控制成肌细胞融合的起始,促使成肌细胞的增殖作用,使单核成肌细胞转变成为多核的肌纤维。因此被认为在MyoD家族中具有中心地位。本文就MyoG基因的结构、定位、作用机制、多态性及其与生产性能的关系等作以下综述。     

Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.

The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.     

一菌双酶法制备L-4-氟苯丙氨酸

利用重组E．coli产天冬氨酸酶和天冬氨酸转氨酶催化生产L-4-氧苯丙氨酸的工艺。实验结果表明最佳转化条件为-37℃,pH值4．5—8．5,菌体与酮酸的质量浓度比为1.5,CTAB的质量分数为0．04％,酮酸的质量浓度11．28g／L,富马酸铵与酮酸的摩尔比为3．0：1．0,添加1mmol／L的Fe^2＋,L-天冬氨酸与酮酸的摩尔比为0．4：1。在最适条件下,经过14h酶转化反应达到平衡,酮酸转化率可达到95％以上,L-4-氟苯丙氨酸得率也可达到80％以上。此法原料简单易得,为L-4-氟苯丙氨酸的制备提供了一种新方法：     

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic structure,expression and evolution of the alfalfa aspartate aminotransferase genes

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.     

The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 A resolution

An X-ray crystallographic study at 4.5 Å resolution has been carried out with triclinic crystals of chicken mitochondrial aspartate aminotransferase.In the electron density map, the enzyme is clearly visible as an isologous α₂-dimer (105 Å × 60 Å × 50 Å) in which the subunits are associated about a molecular 2-fold axis. Each subunit of dimensions 70 Å × 50 Å × 40 Å contains at least seven helices, one of which is about 50 Å long.Difference maps have revealed the positions of the pyridoxyl and the phosphate moieties of the coenzyme as well as the general substrate binding area. The active sites are on opposite sides of the dimer, about 30 Å apart and close to the intersubunit boundary, so that probably both subunits contribute to each active site. An isolated chain segment, passing in front of the active site and ending in contact with the neighbouring subunit is interpreted as one of the chain termini.     

The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases.

The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate diacarboxylic amino acids with similar rate constants. However, eTATase exhibits approximately 10(2)-10(4)-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to alpha-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L-Asp and L-Glu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidino group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acid.     

Glu-50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure.

Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity high-affinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu-50 (Glu-50-->Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme. In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme. However, the Glu-50-->Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steady-state kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50-->Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.     

The structure and function of tRNA-like domain in E. coli tmRNA.

tmRNA has a dual function both as a tRNA and as an mRNA in trans-translation. tmRNA possesses structural elements similar to canonical tRNAs. Our mutation studies show that the tRNA-like structure is crucial for function as an mRNA as well as a tRNA.     

昆虫类免疫球蛋白结构和功能研究进展

类免疫球蛋白是在无脊椎动物体内发现的唯一的免疫球蛋白家族成员,它对鳞翅目昆虫自身的免疫起着重要的作用。已有研究表明：类免疫球蛋白只存在于鳞翅目昆虫体内,有可溶性和不溶性两种存在方式,在昆虫体内分别具有不同的功能。可溶性类免疫球蛋白通过一些酶和蛋白的作用对入侵昆虫的细菌和病毒进行免疫防御,而不溶性类免疫球蛋白（即以膜结合蛋白的形式出现）对细胞和细胞黏着以及病毒和细菌入侵细胞有着延缓作用。