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【求助】一篇文章中材料与方法的翻译
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Materials and methods (1)E. coli flavohemoglobin gene has been cloned essentially as reported by Membrillo-Hernandez et al.[7] and expressed in E. coli under the control of an IPTG inducible promoter by using a PET11 plasmid.(2)The recombinant protein was purified by means of a three-step chromatographic procedure that avoids ammonium sulfate precipitation and yields >5 mg of pure protein per liter of culture.(3)The E. coli crude extract was loaded onto a DEAE 52 column equilibrated with 10 mM Tris^HCl buffer at pH 8.0 containing 0.1 mM EDTA, 1 mM dithiothreitol and 1 mM potassium cyanide. (4)The flavohemoglobin was eluted with an NaCl gradient at 0.18 M salt concentration. (5)The fractions with flavohemoglobin contents higher than 50%, as judged by SDS^PAGE, were pooled, dialyzed against 5 mM phosphate buffer at pH 7.0 and loaded onto a microceramic hydroxylapatite (Bio-Rad, Hercules, CA, USA) column equilibrated with the same buffer. (6)A 5^50 mM phosphate gradient (pH 7.0) was applied and the protein eluted at a buffer concentration of 15 mM.(7) The fractions containing more than 90% flavohemoglobin were loaded directly onto a DEAE-Toyo Pearl (Toso Haas) column equilibrated in 15 mM phosphate buffer and eluted with an NaCl gradient (0^0.25 M). (8)The protein obtained was s98% pure on SDS^PAGE and was fully saturated with flavin cofactor as judged from the ratio (0.27) between the absorption peak of the heme cyanide complex (419 nm) and the oxidized flavin peak (480 nm). 这篇文献的方法有的地方看不懂，不知道该怎么表达才准确，请高手帮忙翻译一下3、5、7、8句话。 非常感谢大家的帮助啦~~~ 可能内容有点多，希望做过蛋白的同学能帮忙看看，再次谢过 |
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(3) DEAE52柱子用10mMTris-HCl 缓冲液（PH8.0,0.1mMEDTA）平衡后，加入E.coli粗体物； (5) 馏分的黄素血红蛋白含量>50%(SDS-PAGE验证),混合，5mM磷酸缓冲液（PH7.0）透析，用相同的缓冲液平衡后上样，柱子（？）； (7)DEAE-TOYO柱用15mM 磷酸缓冲液平衡，上样（黄素血红蛋白高于90%的馏分），NaCl梯度洗脱（0-0.25mM）； (8) SDS-PAGE 电泳后，蛋白得率可达98%，用黄素辅助因子完全饱和，用血红素氰化物和氧化的黄素吸收峰比率（0.27）来判断饱和度 |
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