英语翻译To confirm that the hdcA gene from Staph.capitis IFIJ12 encodes a functional HDC,we expressed this gene in E.coli following the strategy described in the Materials and Methods section,consisting in amplifying the genes by PCR and cloning

英语翻译To confirm that the hdcA gene from Staph.capitis IFIJ12 encodes a functional HDC,we expressed this gene in E.coli following the strategy described in the Materials and Methods section,consisting in amplifying the genes by PCR and cloning
英语翻译
To confirm that the hdcA gene from Staph.capitis IFIJ12 encodes a functional HDC,we expressed this gene in E.coli following the strategy described in the Materials and Methods section,consisting in amplifying the genes by PCR and cloning the products under the control of the T7 RNA polymerase-inducible /10 promoter.
Cell extracts were used to detect the presence of hyperproduced proteins by SDS-PAGE analysis.Control cells containing the pT7-7 vector plasmid alone did not show expression over the 3-h time course analysed,whereas expression of additional 34Æ2-kDa protein was apparent with cells harbouring pAM28 (Fig.2ai).In addition,cell extracts from E.coli JM109(DE3) (pLysS) cells harbouring the recombinant plasmid pAM28 were able to decarboxylate the histidine present in the reaction to histamine,whereas extracts prepared from control cells containing the vector plasmid alone did not.Figure 2(bi) shows a TLC analysis of the enzymatic reaction.Thus,we could prove experimentally that the hdcA gene encodes a functional
HDC.
As the protein was cloned containing a purification poli-His tag,HDC was purified on a His-TrapTM-FF crude chelating column and eluted with a stepwise gradient of imidazole.Highly purified HDC protein was obtained from pAM28 (Fig.2aii).The eluted HDC protein was dialysed to eliminate the imidazole,and checked for HDC activity.TLC analysis demonstrated that highly purified HDC protein was able to decarboxylate histidine to form histamine (Fig.2bii).
The predicted sequence of the HDC was aligned with HDC proteins from Gram-positive bacteria (Fig.S1).As deduced from the HDC alignment,most of the residues implicated in catalysis and substrate binding in the HDC from Lactobacillus 30a (Gallagher et al.1989) are conserved in the Staph.capitis enzyme.However,the residue Ala-260,forming the hydrophobic pocket,is not conserved in the Staph.capitis HDC protein,and a Gly residue is present in its place.
In addition to the wild-type HDC enzymes,a number of mutants that produce partially active or inactive enzymes have been isolated (Recsei and Snell,1982,Van Poelje et al.1990).More interestingly,mutant 3 of HDC from Lactobacillus 30a,which produces a full-length protein that is slowly autoactivated,shows only one amino acid replacement at position 58 (G58A),the Gly amino acid residue is conserved at this position in all HDC,with exception of Staph.capitis HDC with a Asn residue present in its place.An autoactivation assay was performed in order to know if S.capitis HDC follows a similar slow autoactivation.The result shows that along incubation,Staph.capitis HDC seems to be degraded instead to be autocleaved into an α chain (23 kDa) and a β chain (11.5 kDa) (Fig.3).

英语翻译To confirm that the hdcA gene from Staph.capitis IFIJ12 encodes a functional HDC,we expressed this gene in E.coli following the strategy described in the Materials and Methods section,consisting in amplifying the genes by PCR and cloning
为证实hdcA基因编码一种Staph.capitis IFIJ12 HDC功能,我们解释了这种基因在大肠杆菌的策略中描述的方法和材料部分,包括在提高自身基因克隆和产品的控制下polymerase-inducible T7 RNA / 10个推销员.
被用来探测细胞提取物hyperproduced在场的情况下,通过sds - page法分析蛋白质.包含pT7-7矢量控制细胞中质粒表达了单独没有显示的时间历程,而进行保健蛋白表达的额外的34Æ2-kDa与细胞窝藏pAM28是显而易见的.2ai(图).此外,JM109大肠杆菌细胞提取物(DE3)(pLysS)细胞窝藏重组质粒pAM28能够decarboxylate种子的过程中存在的反应,而提取物组从控制细胞中质粒包含矢量独自没有.图二显示bi(TLC)的酶促反应的分析.因此,我们可以证明hdcA基因编码的实验的功能
HDC.
作为蛋白质被克隆poli-His标签包含了净化,在His-TrapTM-FF HDC洁净柱和eluted原油螯与逐步梯度的聚.高纯度HDC蛋白均来自pAM28.2aii(图).这个eluted HDC蛋白质dialysed来消除咪唑、检查HDC活动.薄层色谱分析表明,高纯度的蛋白质能decarboxylate HDC种子形成胺(图.2bii).
序列的预测是符合HDC HDC由革兰阳性细菌蛋白质(图.S1).作为从HDC对齐,大部分的残留在催化和基材与约束的乳酸菌30a HDC从家里等.(1989)被保存在Staph.capitis酶.然而,剩下的Ala-260,形成了狂犬病的口袋,不保存在Staph.capitis HDC蛋白质、Gly残留在它的位置.
此外,许多野生HDC酶活性的突变体产生部分或者非酶被孤立Recsei(1982年),Snell范Poelje罗卓荆.1990).更有趣的是,突变的乳酸菌30a HDC从第三,产生一种完整的蛋白质,慢慢地autoactivated,只说明一个氨基酸置换在位置58例(G58A),Gly氨基酸残基是保存在该职位的所有HDC,除了Staph.HDC capitis与Asn残留在它的位置.一个autoactivation进行化验,为了知道.capitis HDC相似的autoactivation缓慢.结果表明,Staph.capitis沿着孵化、HDC似乎是autocleaved退化而成一个α连锁(23 kDa)和βkDa连锁(5)(图3).