英语翻译不要单纯的用工具翻译啊原文如下MaterialsandmethodsProteinpreparationThenonlabelledanduniformly15N-and15N/13C-labelledCFP-10andESAT-6werepreparedasdescribedpreviously(Renshawetal,2002,2004).Inaddition,13

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英语翻译
不要单纯的用工具翻译啊 原文如下
Materials and methods
Protein preparation
The nonlabelled and uniformly 15N- and 15N/13C-labelled CFP-10
and ESAT-6 were prepared as described previously (Renshaw et al,
2002,2004).In addition,13C/1H HMQC-NOESY spectra were
acquired from samples of the complex in which only the
nonaromatic residues were uniformly 15N/13C labelled.This was
achieved by the preparation of both proteins from Escherichia coli
grown in labelled minimal media supplemented with 50 mg/l of
L-histidine,L-tyrosine,L-phenylalanine and L-tryptophan (Carr
et al,2003).The mixed complexes of labelled CFP-10 bound to
nonlabelled ESAT-6 and vice versa were produced by mixing
equimolar solutions of the purified proteins at room temperature
in 25mM NaH2PO4,100mM NaCl and 0.02% (w/v) NaN3,pH 6.5,
with the individual proteins at a concentration of 5–15 mM.The
complex was concentrated by ultrafiltration to give 0.35 ml NMR
samples containing 0.9–1.5mM CFP-10 .ESAT-6 complex in either a
90% H2O/10% D2O or 100% D2O buffer as appropriate.
Protein corresponding to a truncated variant of CFP-10 lacking
the final 14 C-terminal residues (Asp87–Phe100) was prepared from
a pET28a-based E.coli expression vector,which was produced
using a PCR-based approach,essentially as described previously
(Renshaw et al,2002).Purification of the expressed protein was
carried out in two stages using a 10ml Q-Sepharose column (Renshaw
et al,2002),with truncated CFP-10 eluted from the column in the
50mM NaCl step at pH 8.0 and in the 20mM NaCl step at pH 5.8.
C-terminally truncated ESAT-6 corresponding to residues 1–84 is
produced as a by-product during purification of the full-length
protein.The two species are separated by anion exchange (Renshaw
et al,2002),with the truncated species eluted from the column in
the 100mM NaCl step.
NMR spectroscopy
NMR spectra were acquired at 351C on either an 800MHz Varian
Inova or a 600MHz Bruker Avance spectrometer.The 2D and 3D
spectra recorded to obtain essentially complete sequence-specific
backbone and side-chain assignments for CFP-10 and ESAT-6 in the
complex,and to obtain conformational constraints for structural
calculations were as follows:1H TOCSY and NOESY; 15N/1H HSQC,
TOCSY-HSQC and NOESY-HSQC; 13C/1H HCCH-TOCSY and HMQCNOESY;
and 15N/13C/1H HNCACB,CBCA(CO)NH and HBHA(CBCACO)
NH,as described previously (Renshaw et al,2004).
The 3D NMR data were processed using NMRPipe (Delaglio et al,
1995),with linear prediction used to extend the effective acquisition
times by up to 1.5-fold in F1 and F2 and mild resolution
enhancement applied in all dimensions using a shifted sine-squared
function.Apart from the omission of linear prediction,the 2D
spectra were similarly processed using Varian or Bruker software.
All the spectra were analysed using the program XEASY (Bartels
et al,1995).

Answer 1

材料和方法
蛋白制备
在nonlabelled和统一15N和15N/13C-labelled资格认证- 10
与ESAT - 6准备如前所述（润沙等人,
2002年,2004年）.此外,13C/1H HMQC - NOESY谱光谱
获得从复杂的样品中,只有
nonaromatic残留一律15N/13C标签.这是
实现从大肠杆菌两个蛋白制备
生长在50毫克/升的补充标签基本培养基
L -组氨酸,L -酪氨酸,L -苯丙氨酸和L -色氨酸（卡尔
等,2003）.资格认证的标记物混合- 10必然
nonlabelled杆菌ESAT - 6和反之亦然被混合产生的副
纯化的蛋白质等物质在室温下的解决方案
磷酸二氢钠在25毫米,100毫米和0.02％氯化钠（瓦特/ V）的氮化钠,pH值6.5,
与在5-15毫米的单个蛋白浓度.那个
复杂集中超滤给0.35毫升核磁共振
样本含有0.9 - 1.5mm的资格认证10.杆菌ESAT - 6无论是复杂的
90％H2O/10％重水或100％的重水适当的缓冲区.
蛋白质相应的资格认证截断变异- 10缺乏
最后的14 C -末端残基（Asp87 - Phe100）的制备
1 pET28a中的大肠杆菌表达载体,生产的
采用PCR技术为基础的方法,基本上如前所述
（润沙等人,2002年）.纯化的表达产物
通过两个阶段,用10毫升调Q Sepharose柱（伦肖
等人,2002年）,与截断资格认证,10列中洗脱
50毫米氯化钠步骤在pH 8.0和20毫米在pH 5.8氯化钠的一步.
ç -末期截断杆菌ESAT - 6对应的残留1-84
产生作为副产品期间全长净化产品
蛋白质.这两个品种的分隔离子交换（伦肖
等人,2002年）,同种洗脱截断从列
在100毫米氯化钠一步.
核磁共振光谱
核磁共振光谱,在351C上获得或800MHz的瓦里安
伊诺瓦或600MHz的布鲁克Avance系列光谱仪.二维和三维
光谱记录获得基本完成序列特异性
骨干和侧链任务的资格认证- 10与ESAT - 6
复杂,获取结构的构象限制
计算如下：上半年TOCSY和NOESY谱; 15N/1H HSQC,
TOCSY - HSQC和NOESY谱,HSQC,13C/1H胆管癌- TOCSY和HMQCNOESY;
和15N/13C/1H HNCACB,CBCA（CO）的NH和HBHA（CBCACO）
新罕布什尔州如前所述（润沙等人,2004年）.
三维NMR数据进行了处理使用NMRPipe（Delaglio等,
1995年）,与线性预测用于扩展有效的收购
高达1.5倍倍F1和F2和温和的决议
在所有使用方面的应用转向加强正弦平方
功能.除了线性预测,二维遗漏
光谱也同样处理或使用瓦里安布鲁克软件.
所有的光谱进行了分析,使用该程序XEASY（巴特尔斯
等,1995）.