Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy

DENG Tong,ZHOU Hai-sheng,WU Jian-ping,YANG Li-rong

China Biotechnology ›› 2020, Vol. 40 ›› Issue (8) : 24-32.

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China Biotechnology ›› 2020, Vol. 40 ›› Issue (8) : 24-32. DOI: 10.13523/j.cb.2005006

Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy

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Abstract

Objectives: Many of biocatalytic redox reactions which are widely used in the production of chiral chemicals involve the regeneration of the coenzyme NADPH in situ. Alcohol dehydrogenases that regenerate NADPH with isopropanol as substrate have the advantages of high specific activity and easy separation of byproduct acetone, attracting more and more attention. Therefore, an alcohol dehydrogenase from Clostridium beijerinckii, namely CbADH, was chosen as the research object for its more considerable specific activity and the most applicable potentiality within present literatures. To solve the problem of poor soluble expression of CbADH in E. coli genetically engineered strains and the consequent enzyme activity as low as 2.31 U / mg DCW, the following studies were carried out. Methods: Firstly, different chaperone proteins were expressed by inducible plasmids to increase the soluble expression level of CbADH, and the results showed that molecular chaperone GroES-GroEL significantly improved the soluble expression of CbADH by 3.57 times more than the original strain, with enzyme activity of 11.18 U/mg DCW which is 4.83 times more than the original strain. Secondly, three other different GroES-GroEL expression strategies were examined: pET-28a(+) single plasmid co-expression, genomic enhancing expression of chaperone, and constitutive-pGro7/pET-28a(+) dual plasmid co-expression. Results: The results indicated that the constitutive-pGro7/pET-28a(+) dual plasmid co-expression strategy had the best effect which improved the soluble expression of CbADH by 8.07 times more than the oringinal strain, with a CbADH activity of 21.79 U/mg DCW, which was 9.43 times higher than the oringinal strain. Conclusions: This study not only lays the foundation for the industrial application of CbADH but also provides a reference for heterologous soluble protein expression.

Key words

Alcohol dehydrogenase / Soluble heteroexpression / Chaperone / NADPH

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Tong DENG, Hai-sheng ZHOU, Jian-ping WU, et al. Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy[J]. China Biotechnology, 2020, 40(8): 24-32 https://doi.org/10.13523/j.cb.2005006
氧化还原酶广泛应用于手性化合物的制备[1],其催化反应过程需要NADH或NADPH作为辅酶。近些年在针对非天然产品的制备研究中,发现大多数具有高立体选择性和高催化活力的氧化还原酶都是NADPH依赖型[2]。由于NADPH自身不稳定、价格昂贵且需要等当量消耗[3],需要通过辅酶再生酶的催化进行原位再生体。目前的研究中,主要使用葡萄糖脱氢酶进行NADPH的再生[4,5],但由于副产物葡萄糖酸的产生,导致过程原子经济性差、产品精制成本高、环保压力大,已不满足现今的工业要求。而醇脱氢酶以异丙醇作为原料再生NADPH,具有原子经济性高、副产物丙酮易于分离等优势,具有极高工业应用潜力。文献报道的醇脱氢酶多数是NADH依赖型,可用于原位再生NADPH的醇脱氢酶种类较少。催化NADPH再生的比活力较高的醇脱氢酶有来源于Thermococcus guaymasensis strain DSM 11 113的TgADH[6]以及来源于Methanogenium thermophilum的MtADH[7],催化异丙醇再生NADPH的比酶活分别为245 U/mg蛋白(35℃)和176 U/mg蛋白(40℃);但这两种酶蛋白稳定性差,室温条件下在空气中易失活,不利于工业化应用。此外,热稳定性较好的NADPH依赖型醇脱氢酶有来源于Thermoanaerobium brockii的TbADH[8]以及来源于Entamoeba histoytica的EhADH[9],这两种酶的 T5060min (保温60min后酶活损失一半的温度)温度分别为93.8℃、77.5℃,但二者的比酶活分别只有59U/mg蛋白(40℃)和29U/mg蛋白(25℃);此外,来源于嗜热菌株Thermoanaerobacter ethanolicus的醇脱氢酶TeADH也具备高热稳定性,但其比活仅有60U/mg 蛋白(60℃)[10];而来源于Clostridium beijerinckii的CbADH在25℃下比酶活达到了140 U/mg蛋白[11],且其 T5060min (保温60min后酶活损失一半的温度)也有64.5℃,CbADH兼具高比活和高稳定性,是目前最为合适的可用于辅酶NADPH再生的醇脱氢酶。
关于CbADH的研究主要集中在对其晶体结构解析[12]与热稳定性改造[13]。如,Bogin等[14]通过构建CbADH与TbADH的嵌合体,探索影响醇脱氢酶的热稳定性的结构因素;Goihberg等[15]用定点突变技术将CbADH的100位点的谷氨酰胺替换为脯氨酸,使其 T5060min 提高了8℃。然而,对于CbADH异源表达的研究则较少,Peretz等[11]和Ismaiel等[16]将CbADH克隆、测序并构建到pUC18质粒上,转化进入E.coli DH5α菌株中进行异源表达,CbADH的发酵液酶活仅为1.73 U/ml。由于缺乏针对CbADH的高效、简便的异源表达策略,限制了CbADH的工业应用。为使CbADH高水平表达,本研究首先构建pET-28a(+)/CbADH质粒,并在大肠杆菌表达宿主E.coli BL21(DE3)上重组表达CbADH。其次,为了解决CbADH高表达可能产生的可溶性差的问题,引入分子伴侣与目标酶共表达,并优化共表达策略,以获得CbADH的高产菌株。

1 材料与方法

1.1 材料

1.1.1 菌株和质粒 E.coli BL21(DE3)感受态细胞由本实验室保藏;pCas,pTargetF质粒以及5种表达分子伴侣蛋白的质粒(pG-KJE8、pGro7、pKJE7、pG-tf2、pTf16)购自宝生物工程有限公司(大连);载有CbADH基因(NCBI登录号WP_077844196.1)的pET-28a(+)质粒、引物合成与测序均由北京擎科新业生物技术有限公司完成。
1.1.2 主要试剂 DNA marker、DNA Polymerase、Protein SDS PAGE Loading Buffer等购自宝生物工程有限公司(大连);卡那霉素(Kan)、壮观霉素(Spe)、氯霉素(Cm)、异丙基硫代-β-D-半乳糖苷(IPTG)、四环素等试剂购自北京鼎国生物技术有限公司;质粒DNA提取试剂盒、DNA胶回收试剂盒均购自杭州Axygen公司;Protein Ladder购自美国 Thermo Fisher Scientific 公司;辅酶NADP+购自邦泰生物工程(深圳)有限公司;其他常用试剂均购自国药集团化学试剂有限公司。
1.1.3 主要仪器 TU-1810PC型紫外可见分光光度计,北京普析通用仪器有限公司;Thermomixer型恒温金属浴,德国Eppendorf公司;JY92-II型超声波细胞粉碎机,宁波新芝生物科技有限公司;GL-2050M型高速冷冻离心机,上海卢湘仪离心机有限公司;A200型PCR仪,杭州朗基科学仪器有限公司;PowerPac Basic型电泳仪,BIO-RAD (美国)公司。

1.2 方法

1.2.1 CbADH表达菌株构建 CbADH全基因合成序列和pET-28a(+)/CbADH质粒载体构建由北京擎科新业生物技术有限公司完成。将pET-28a(+)/CbADH质粒载体转化E.coli BL21(DE3) 感受态细胞,得到E.coli BL21(DE3)-pET-28a(+)/CbADH重组菌株。
1.2.2 分子伴侣与CbADH共表达菌株的构建与培养 把E.coli BL21(DE3)-pET-28a(+)/CbADH重组菌株制备成感受态细胞,分别将5种分子伴侣质粒(表1)转化进入其中,将这些菌株依次命名为A,B,C,D,E。在双抗LB固体培养基(含50μg /ml Kan和20μg /ml Cm)上挑取单克隆菌落进行PCR鉴定,并转移到5ml LB液体培养基(含50μg/ml Kan和20μg/ml Cm)中,在摇床中37℃、200r/min扩大培养12h,保存菌种。
Table 1 Information of molecular chaperone plasmid

表1 分子伴侣质粒信息

分子伴侣质粒 伴侣蛋白 启动子 诱导剂 抗性
pG-KJE8 DnaK-DnaJ-GrpE-GroES-GroEL araB Pzt-1 0.5g/ml L-Arabinose+5ng/ml Tetracyclin Cm
pGro7 GroES-GroEL araB 0.5g/ml L-Arabinose Cm
pKJE7 DnaK-DnaJ-GrpE araB 0.5g/ml L-Arabinose Cm
pG-Tf2 GroES-GroEL-Tf Pzt-1 5ng/ml Tetracyclin Cm
pTf16 Tf araB 0.5g/ml L-Arabinose Cm
1.2.3 pET单质粒共表达菌株的构建 基于pET-28a(+)质粒骨架,通过SD序列的引入,在一个T7启动子下实现两个蛋白的共表达,构建了F和G两个共表达质粒,如图1所示,质粒F与质粒G之间主要区别是CbADH与GroES-GroEL的构建位置相反。此外,在质粒F与G的基础上用全质粒PCR扩增引入第二个T7启动子,构建了H和I两个共表达载体,如图1所示。具体步骤参照《分子克隆手册》进行。将质粒F,G,H,I分别转化E.coli BL21(DE3)感受态细胞,即得到单质粒共表达分子伴侣与CbADH的4种菌株F,G,H,I。
Fig.1 Expression region structure of the co-expression plasmids

图1 共表达质粒的表达区域结构示意图

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1.2.4 基因组强化表达GroES-GroEL菌株的构建 用CRISPR基因组编辑技术[17]将GroES-GroEL表达基因groESL前的σ32启动子替换为强度更高的三种组成型启动子:J23119、J23100、J23114(表2),以实现基因组强化表达GroES-GroEL,基因操作示意图见图2。获得基因组编辑后的菌株并将它们分别制备为感受态细胞,将pET-28a(+)/CbADH质粒转化进入其中,分别得到基因组强化表达GroES-GroEL的菌株J、K、L。
Table 2 Information of constituent promoters

表2 组成型启动子信息(① 组成型启动子具体信息来源于http://parts.igem.org/Promoters/Catalog/Anderson)

启动子名称 序列 相对强度
J23119 ttgacagctagctcagtcctaggtataatgctagc n/a
J23100 ttgacggctagctcagtcctaggtacagtgctagc 1
J23102 ttgacagctagctcagtcctaggtactgtgctagc 0.86
J23104 ttgacagctagctcagtcctaggtattgtgctagc 0.72
J23108 ctgacagctagctcagtcctaggtataatgctagc 0.51
J23110 tttacggctagctcagtcctaggtacaatgctagc 0.33
J23114 tttatggctagctcagtcctaggtacaatgctagc 0.10
Fig.2 Schematic diagram of CRISPR genome editing modified promoter

图2 CRISPR基因组编辑示意图

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1.2.5 组成型pGro7和pET双质粒共表达菌株的构建 以pGro7质粒为模板,分别以23119-1/2、23100-1/2、23102-1/2、23104-1/2、23108-1/2、23110-1/2、23114-1/2为引物,以全质粒PCR扩增、将araBo|p启动子替换为组成型启动子(组成型启动子信息见表2),转化到E.coli BL21(DE3)-pET-28a(+)/CbADH菌株感受态细胞中,即完成启动子替换的双质粒菌株的构建,将这些菌株分别命名为M,N,O,P,Q,R,S。
1.2.6 摇瓶发酵产酶 将单克隆菌落挑取至20 ml LB液体培养基中,在摇床中37℃、200r/min培养12h后转入230ml LB培养基中,在摇床中37℃、200r/min培养3h,加入250μl 0.5mol/L IPTG诱导剂,降温到25℃继续培养16h。其中1.2.2中的共表达菌株需进行分子伴侣与CbADH的分级诱导,转接时需在培养基中按表1加入相应分子伴侣诱导物。
1.2.7 菌体浓度测定 从发酵液中取100μl,用去离子水稀释10倍,用分光光度计在600nm处测定吸光值,菌体浓度(OD600)=10×600nm处吸光值。
1.2.8 细胞干重测定 将发酵液置于100ml 离心管中,4℃、12 000r/min离心5min,倒去上清液,用0.1mol/L pH 7.5的PBS缓冲液在4℃、12 000r/min下离心清洗沉淀3次,获得湿菌体,置于-80℃冰箱中冷冻保存。取一部分冷冻后的菌体,称重后转移到75℃烘箱中烘干。烘干至恒重后即为细胞干重。
1.2.9 醇脱氢酶酶活测定 称取一部分湿菌体,置于100ml离心管中,按1 g/100ml的比例加入0.1 mol/L pH 7.5的PBS缓冲液,重悬细胞,在冰水浴中超声破碎(功率400 W,超声次数100次,每次超声3 s,间隔7s),获得粗酶液。取100 μl粗酶液迅速加入900μl反应液(800μl 62.5mmol/L的异丙醇溶液(pH 7.5),100μl 10mmol/L NADP+溶液,混合后在35℃金属浴中预热10min)中,将总计1ml的反应体系在340nm波长下光度扫描60s,以时间为横坐标、吸光值为纵坐标线性拟合,得到斜率值,再结合NADPH的摩尔消光系数(6220L/mol/cm)计算酶活。酶活定义为:在所述条件下,每分钟生成1μmol NADPH所需的酶量。在本研究中,酶活单位为U/mg干细胞重(DCW)。
1.2.10 SDS-PAGE蛋白电泳 取1.2.9节获得的粗酶液中50μl作为全细胞样品;另取同样的粗酶液1ml,12 000r/min离心10min后,取50μl作为上清样品(可溶蛋白样品);除上清后的沉淀,加入1ml去离子水重悬,取50μl作为沉淀样品。在以上样品中分别加入10μl 4×Protein SDS PAGE Loading Buffer,混匀后于99℃变形10 min,上样进行SDS-PAGE电泳。
1.2.11 蛋白质灰度分析 使用ImageJ软件对蛋白电泳结果进行灰度分析,以确认各个蛋白大致的表达量。将蛋白电泳结果图导入到ImageJ软件中,进行参数设置:将Type调整为8-bit;在substract background中选择light background选项,将rolling ball radius调整为50.0 pixels;在set scale中将unit of length设置为pixels。使用invert功能,将图片转化为黑底。使用freehand selections将要进行灰度分析的区域精准选定,点击Measure进行测量。其中integrated density即为该区域的灰度值。由于选定区域时存在一定的误差,因此选定5次取平均值用于后续工作。

2 结果与分析

2.1 CbADH在E.coli BL21(DE3)中的重组表达

本实验首先构建了pET-28a(+)/CbADH质粒,导入大肠杆菌表达宿主E.coli BL21(DE3),得到重组表达CbADH的菌株CbADH-WT。SDS-PAGE电泳结果(图3)显示CbADH在常规诱导表达条件下出现了严重的包涵体,仅获得少量的可溶性蛋白;酶活测定结果(表3)为2.31 U/mg DCW(约为2.91 U/ml),这与CbADH在E.coli DH5α菌株中的酶活(1.73U/ml)相近,表明CbADH的异源可溶性表达存在一定困难。
Fig.3 SDS-PAGE analysis of molecular chaperones co-expression strains

Lane M:molecular weight marker;Lane W:whole cell protein; Lane S:supernatant;Lane P:precipitation

图3 分子伴侣共表达菌株的SDS-PAGE分析

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Table 3 CbADH activity of different strategies

表3 不同策略的CbADH发酵液酶活测定结果

采用策略 菌株名称 OD600 酶活(U/mg DCW)
出发菌株 CbADH-WT 5.91±0.34 2.31±0.3
分子伴侣共表达 A 6.24±0.38 11.18±0.96
B 5.27±0.2 3.61±0.21
C 5.17±0.31 3.98±0.14
D 7.19±0.24 4.17±0.35
E 5.97±0.23 2.88±0.09
单质粒共表达 F 5.4±0.24 10±0.35
G 5.23±0.32 7.89±0.46
H 4.88±0.28 8.02±0.43
I 5.09±0.19 12.99±0.89
基因组强化表达 J 5.23±0.35 9.13±0.31
K 5.31±0.37 7.26±0.18
L 5.28±0.25 2.3±0.24
组成型双质粒共表达 M 5.08±0.41 21.79±1.1
N 5.44±0.24 17.74±0.92
O 5.43±0.32 16.8±0.42
P 5.56±0.21 15.57±0.52
Q 5.79±0.13 15.08±0.46
R 5.55±0.32 14.82±0.32
S 5.32±0.26 12.35±0.67

2.2 不同的分子伴侣蛋白对CbADH可溶性表达的影响

本实验首先通过简单的商业化质粒、引入不同伴侣蛋白与CbADH共表达,考察分子伴侣蛋白过表达能否提高CbADH的可溶性表达水平。所选分子伴侣表达体系为:Strain A-pGro7/GroES-GroEL、Strain B-pKJE7/DnaK-DnaJ-GrpE、Strain C-pG-KJE8/DnaK-DnaJ-GrpE-GroES-GroEL、Strain D-pG-Tf2/GroES-GroEL-Tf、Strain E-pTf16/Tf(菌株-质粒/分子伴侣)。各个菌株酶活测定与蛋白电泳结果分别如表3图3所示。
结果显示表达分子伴侣蛋白GroES-GroEL的菌株A的酶活最高,为11.18 U/mg DCW,是出发菌株的4.83倍,其CbADH的可溶性部分明显高于出发菌株。该结果表明分子伴侣蛋白GroES-GroEL可以显著提高CbADH的可溶性表达,而其他分子伴侣蛋白对CbADH的可溶性表达影响不明显。根据分子伴侣蛋白的作用机制,分子伴侣蛋白Tf与DnaK-DnaJ-GrpE作用于蛋白折叠初始阶段、通过保护初生多肽链免于错误聚集[18,19],而GroES-GroEL主要协助部分折叠态的蛋白克服动力学障碍、正确折叠为其天然状态[20,21,22]。由此推断CbADH蛋白折叠问题不在于早期的错误聚集,而在于其部分折叠态跨越动力学障碍存在困难。

2.3 pET单质粒共表达策略提高CbADH的可溶性表达

鉴于菌株A表达CbADH仍存在明显的包涵体,为获得更高的CbADH酶活,需要进一步提高GroES-GroEL的表达量。因此,构建了两类pET单质粒共表达策略,其中一类为两种蛋白均在同一T7启动子下表达,另一类为两种蛋白前各有一个T7启动子;分别调换两种蛋白的基因顺序得到4种不同单质粒共表达策略,以获得GroES-GroEL与CbADH不同的表达量和比例。酶活测定与蛋白电泳结果分别如表3图4所示。
Fig.4 SDS-PAGE analysis of pET single plasmid co-expression strains

Lane M: molecular weight marker;Lane W:whole cell protein; Lane S: supernatant;LaneP:precipitation

图4 pET单质粒共表达菌株的SDS-PAGE分析

Full size|PPT slide

结果显示,单质粒共表达策略实现了GroES-GroEL的增量表达,且CbADH的可溶性表达随着GroES-GroEL的表达量提高而提高。但GroES-GroEL的增量表达导致了CbADH总表达量相比出发菌株和菌株A明显降低,使CbADH的酶活提升不大。此外,菌株H与I同为双启动子共表达,差别在于两个基因的顺序不同。菌株H的GroES-GroEL的表达量较高、CbADH的表达量较低;而菌株I则相反,这是由于第二个T7启动子之后的基因有可能会被转录两次。

2.4 基因组强化表达GroES-GroEL策略提高CbADH的可溶性表达

单质粒共表达策略使分子伴侣蛋白表达量过高,导致目标酶CbADH的蛋白表达量下降。因此尝试了下调GroES-GroEL的表达量的策略以获得更高的CbADH酶活。通过CRISPR基因组编辑技术,将E.coli BL21(DE3)基因组上的GroES-GroEL表达基因groESL前的启动子替换为强度更高的三种启动子:J23119、J23100、J23114,分别得到菌株J、K、L,以期实现基因组强化表达GroES-GroEL。酶活测定与蛋白电泳结果分别如表3图5所示。结果显示菌株J,K实现了基因组强化表达GroES-GroEL。虽然其伴侣蛋白表达量和CbADH酶活显著高于出发菌株,但低于菌株A,表明该策略的伴侣蛋白表达量过低,未能有效解决CbADH可溶性表达的问题。
Fig.5 SDS-PAGE analysis of GroES-GroEL genomic over-expression strains

Lane M: molecular weight marker; Lane W: whole cell protein; Lane S: supernatant; Lane P: precipitation

图5 基因组强化表达GroES-GroEL的SDS-PAGE分析

Full size|PPT slide

2.5 组成型pGro7和pET双质粒共表达策略提高CbADH的可溶性表达

由于GroES-GroEL蛋白表达在单质粒共表达策略中过量,而在基因组强化表达策略中过低。为获得GroES-GroEL表达量适中,CbADH酶活最高的菌株,选用了7种强度呈梯度的组成型启动子、替换质粒pGro7上的诱导型启动子araBo|p,得到7种组成型质粒pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达菌株。酶活测定与蛋白电泳结果分别如表3图6所示。
Fig.6 SDS-PAGE analysis of constitutive pGro7/GroES-GroEL and pET-28a(+)/CbADH dual plasmid co-expressed strains

Lane M: molecular weight marker;Lane W:whole cell protein;Lane S:supernatant;Lane P:precipitation

图6 组成型改造pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达菌株SDS-PAGE分析

Full size|PPT slide

结果显示,组成型质粒pGro7和pET-28a(+)双质粒共表达菌株的CbADH酶活与可溶性表达均随着启动子强度提高而提高。其中,菌株M的酶活达到了21.79U/mg DCW,是菌株A菌株的约1.95倍,是出发菌株的约9.43倍,通过灰度分析,其可溶性表达较出发菌株提高了8.07倍。菌株M~S的分子伴侣表达量处于单质粒共表达策略与基因组强化策略之间,而酶活高于这两种策略。

2.6 分子伴侣GroES-GroEL的表达量与CbADH酶活的关系

为探究分子伴侣GroES-GroEL的表达量与CbADH酶活的关系,通过ImageJ软件对蛋白电泳结果进行灰度分析获得全细胞蛋白中GroEL与CbADH的比例,以此为横坐标,以CbADH的酶活为纵坐标对各个菌株的酶活和蛋白表达结果进行作图,如图7所示。
Fig.7 Relationship between GroES-GroEL expression and CbADH activity

图7 GroEL与CbADH表达量比例同CbADH酶活的关系

Full size|PPT slide

图7的结果表明,在GroEL占比低时,CbADH酶活随着GroEL占比的提高而上升;当GroEL占比超过一定值时,CbADH酶活随着GroEL占比的提高而下降。在本研究采用的4种表达策略所获得的菌株中,组成型改造pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达的菌株M的CbADH酶活最高,其GroEL/CbADH的值为0.71,这可能是伴侣蛋白GroES-GroEL与CbADH的最佳表达量之比。

3 结论

通过引入分子伴侣提高了NADPH依赖型醇脱氢酶CbADH的可溶性表达,发现分子伴侣GroES-GroEL对CbADH的可溶性表达影响显著。此外,通过四种不同的GroES-GroEL与CbADH共表达策略,获得了分子伴侣蛋白与目标酶蛋白不同的表达量和表达比例,结果发现分子伴侣GroES-GroEL的表达量过高和过低均不利于提高目标酶CbADH酶活。在本实验所构建的菌株中,组成型改造pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达菌株M的酶活最高,其GroEL和CbADH的表达量比值为0.71,CbADH的可溶性表达较出发菌株提高了8.07倍,酶活达到了21.79U/mg DCW,是出发菌株的约9.43倍。虽然共表达分子伴侣策略对提高CbADH可溶性表达的效果明显,但分子伴侣的表达或多或少会占用细胞用于表达目标酶蛋白的资源,导致目标酶表达量减少,因此,未来的研究可以直接对CbADH进行蛋白质工程改造,避免目标酶表达量下降的同时提高其可溶性表达,以进一步提升CbADH的酶活。

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A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16-20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.

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