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学者姓名：林海英
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Abstract ：
1,3-butanediol is an important diol used in chemical synthesis, food and medicine, daily chemical production, and more. In this study, Escherichia coli BL21 (DE3) was used as the host bacterium. The 1,3-butanediol metabolic synthesis pathway was constructed using acetyl-CoA acetyltransferase PhaA, NAD (P) H-dependent acetyl-CoA reductase PhaBl and butaldehyde dehydrogenase Bld. On this basis, the effects of aldehyde-ketone reductase PA1127 and NAD kinase were explored. The effects of NADK on the metabolic production of 1,3-butanediol in recombinant strains were studied, and the culture conditions of 1,3-butanediol producing recombinant strains were optimized in order to improve the production of 1,3-butanediol in the recombinant strains. The recombinant plasmid was constructed in the form of multi-plasmid co-transformation system and multi-cis-trans subsystem, and the recombinant strain was obtained by transformation. After the conversion experiment or fermentation, glucose consumption was determined by the DNS method, bacterial volume and NADPH levels were compared by light absorption value, and 1,3-butanediol and by-products were quantitatively analyzed by GC and HPLC. The 1,3-butanediol metabolic synthesis pathway consisting of PhaA, PhaBl and Bld was successfully constructed, and the conversion of glucose to 1,3-butanediol was realized. Overexpression of NADK improved the ability of recombinant strains to synthesize 1,3-butanediol, but increased the yield of acetic acid, lactic acid, ethanol and other by-products. By optimizing culture conditions and medium components during fermentation, the yield of 1,3-butanediol was increased to 0.531 g/L. This conclusion provides a theoretical basis for the study of 1,3-butanediol biosynthesis. © 2024 China Biotechnology Press. All rights reserved.
Keyword ：
1,3-Butanediol 1,3-Butanediol Aldehyde-ketone reductase Aldehyde-ketone reductase Metabolic pathways Metabolic pathways NADK NADK PA1127 PA1127
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| GB/T 7714 | Lin, H. , Yu, Y. , Chen, Y. et al. Improvement of 1,3-Butanediol Production by Engineered Escherichia coli; [1,3-丁二醇合成细胞的构建及优化] [J]. | China Biotechnology , 2024 , 44 (11) : 30-38 . |
| MLA | Lin, H. et al. "Improvement of 1,3-Butanediol Production by Engineered Escherichia coli; [1,3-丁二醇合成细胞的构建及优化]" . | China Biotechnology 44 . 11 (2024) : 30-38 . |
| APA | Lin, H. , Yu, Y. , Chen, Y. , Zhang, M. . Improvement of 1,3-Butanediol Production by Engineered Escherichia coli; [1,3-丁二醇合成细胞的构建及优化] . | China Biotechnology , 2024 , 44 (11) , 30-38 . |
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1,3-丁二醇(1,3-butanediol)是应用于化工合成、食品药品、日化生产等领域的重要二元醇,以大肠杆菌BL21(DE3)为宿主菌,利用乙酰辅酶A乙酰基转移酶PhaA、NAD(P)H依赖型乙酰乙酰辅酶A还原酶PhaB1、丁醛脱氢酶Bld构建1,3-丁二醇代谢合成途径,在此基础上探究醛酮还原酶PA1127及NAD激酶(NAD kinase,NADK)对重组菌株代谢生产1,3-丁二醇的影响,并对产1,3-丁二醇重组菌株进行培养条件优化,以期提高重组菌株的1,3-丁二醇产量.采用多质粒共转化系统和多顺反子系统两种形式构建重组质粒,转化获得多酶共表达重组菌株.在转化试验或发酵结束后,利用丹磺酰法(DNS法)检测葡萄糖消耗量、通过吸光值检测比较菌体量和NADPH水平、采用GC和HPLC对1,3-丁二醇和副产物进行定量分析.成功构建由PhaA、PhaB1、Bld组成的1,3-丁二醇代谢合成途径,并实现葡萄糖向1,3-丁二醇的转化;NADK过表达使重组菌株合成1,3-丁二醇的能力提高,同时使乙酸、乳酸、乙醇等副产物产量增加,发酵过程培养条件及培养基组分的优化可提高重组菌株1,3-丁二醇产量达0.531 g/L.此结论可为1,3-丁二醇生物合成研究提供理论依据.
Keyword ：
1 1 3-丁二醇 3-丁二醇 NAD激酶 NAD激酶 代谢途径 代谢途径 醛/酮还原酶PA1127 醛/酮还原酶PA1127
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| GB/T 7714 | 林海英 , 于艺洁 , 陈怡平 et al. 1,3-丁二醇合成细胞的构建及优化 [J]. | 中国生物工程杂志 , 2024 , 44 (11) : 30-38 . |
| MLA | 林海英 et al. "1,3-丁二醇合成细胞的构建及优化" . | 中国生物工程杂志 44 . 11 (2024) : 30-38 . |
| APA | 林海英 , 于艺洁 , 陈怡平 , 张曼 . 1,3-丁二醇合成细胞的构建及优化 . | 中国生物工程杂志 , 2024 , 44 (11) , 30-38 . |
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Objective: Chemical coupling agent and polylysine (PL) were used to conjugate goat anti-rabbit IgG and horseradish peroxidase (HRP) to increase the number of HRP connections on antibodies to amplify the sensitivity of immune detection, and the sensitivity was detected and compared through immunodetection applications, which is of great significance for subsequent immune diagnosis and biological research. Methods: Tylthioacetate (SATA) and Traut’s were optimized for the polymer HRP-PL, the feed ratio of Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) was optimized with goat anti-rabbit IgG and HRP-PL, and the reaction ratio of the two was optimized. The conjugated IgG-PL-HRP and commercial secondary antibody were detected by immunoblot, ELISA and immunohistochemistry, respectively, and the amplification of conjugated IgG-PL-HRP was calculated. Results: When the molar ratio of PL to HRP was 1∶5, the molar ratio of HRP-PL to Traut’s was 1∶15, the molar ratio of goat anti-rabbit IgG to Sulfo-SMCC was 1∶30, the molar ratio of goat anti-rabbit IgG to HRP-PL was 1∶10, the reaction efficiency was high. In the immune spot test, the minimum detection limits of commercial secondary antibody and conjugate IgG-PL-HRP were 2.5 μg and 312.5 ng, respectively, and the maximum dilution times were 50 and 100 times, respectively. In ELISA experiment, the maximum dilution times were 5 000 and 20 000, times respectively. In the immunohistochemical experiment, the detection specificity and intensity of conjugated IgG-PL-HRP were higher than those of commercial secondary antibody. Conclusion: The primary amplification conjugate IgG-PL-HRP was successfully synthesized and its immune detection signal amplification was about 3 ~ 7 times that of the commercial secondary antibody. It is of great significance to the following immunodiagnosis and biological research. © 2023, China Biotechnology Press. All rights reserved.
Keyword ：
Antibody conjugate Antibody conjugate Horseradish peroxidase Horseradish peroxidase Polylysine Polylysine Signal amplification Signal amplification
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| GB/T 7714 | Shen, Y. , Yao, B.-Y. , Yang, R.-N. et al. Preparation and application of immunoassay signal amplification antibody conjugates [J]. | China Biotechnology , 2023 , 43 (1) : 42-49 . |
| MLA | Shen, Y. et al. "Preparation and application of immunoassay signal amplification antibody conjugates" . | China Biotechnology 43 . 1 (2023) : 42-49 . |
| APA | Shen, Y. , Yao, B.-Y. , Yang, R.-N. , Kang, W.-B. , Lin, H.-Y. . Preparation and application of immunoassay signal amplification antibody conjugates . | China Biotechnology , 2023 , 43 (1) , 42-49 . |
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目的:以聚赖氨酸(polylysine,PL)为骨架提高辣根过氧化物酶(horseradish peroxidase,HRP)与羊抗兔IgG的连接数量,比较几种化学偶联剂的偶联效果,通过免疫检测技术对其灵敏度进行检测和比较.方法:对HRP与PL、聚合物HRP-PL与N-琥珀酰-S-乙酰乙酸(N-succinimidyl-S-acetylthioacetate,SATA)和2-亚氨基硫烷(Traut's)两种试剂、羊抗兔IgG与琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯[succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,Sulfo-SMCC]、活化后羊抗兔IgG及HRP-PL进行摩尔比的优化;对偶联物IgG-PL-HRP及商品化二抗分别进行斑点免疫印迹、ELISA和免疫组化,并计算偶联物IgG-PL-HRP的检测放大倍数.结果:当PL与HRP摩尔比为1∶5,HRP-PL与Traut's摩尔比为1∶15,羊抗兔IgG与Sulfo-SMCC摩尔比为1∶ 30,羊抗兔IgG与HRP-PL摩尔比为1:10时,反应效率较高;商品化二抗及偶联物IgG-PL-HRP在斑点免疫印迹实验中的最低检测限分别为2.5μg和312.5 ng,最大稀释倍数分别为50和100倍;在ELISA实验中的最大稀释倍数分别为5 000和20 000倍;在免疫组化实验中偶联物IgG-PL-HRP的检测特异性及强度均大于商品化二抗.结论:成功合成抗体偶联物IgG-PL-HRP,且免疫检测信号放大倍数为商品化二抗的3～7倍,这对后续免疫诊断及生物学的研究具有重要意义.
Keyword ：
信号放大 信号放大 抗体偶联物 抗体偶联物 聚赖氨酸 聚赖氨酸 辣根过氧化物酶 辣根过氧化物酶
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| GB/T 7714 | 沈妍 , 姚玢妍 , 杨若楠 et al. 免疫检测信号放大抗体偶联物的制备及应用 [J]. | 中国生物工程杂志 , 2023 , 43 (1) : 42-49 . |
| MLA | 沈妍 et al. "免疫检测信号放大抗体偶联物的制备及应用" . | 中国生物工程杂志 43 . 1 (2023) : 42-49 . |
| APA | 沈妍 , 姚玢妍 , 杨若楠 , 康文斌 , 林海英 . 免疫检测信号放大抗体偶联物的制备及应用 . | 中国生物工程杂志 , 2023 , 43 (1) , 42-49 . |
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目的：构建靶向磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)的嵌合抗原受体(chimeric antigen receptor, CAR)及靶向上皮细胞黏附分子(epithelial cell adhesion molecule, EPCAM)嵌合抗原共刺激受体(chimeric antigen costimulatory receptor, CCR)共修饰的T细胞，并对其进行体外活性评估。方法：将EPCAM-CCR和GPC3-CAR基因片段克隆入慢病毒载体质粒，酶切、PCR和测序鉴定CCR+CAR pCDH重组载体。分离、激活和扩增人T细胞，利用慢病毒感染并筛选能够稳定表达该组合型嵌合抗原受体人T细胞。通过Western blot、流式细胞术(flow cytometry, FCM)验证CCR+CAR T细胞中CCR+CAR的表达，ELISA检测细胞因子IL-2、INF-γ、IL-4的分泌。结果：成功构建CCR+CAR pCDH慢病毒重组载体。成功分离、激活并扩增人T细胞。CCR+CAR pCDH慢病毒成功感染人T细胞，RT-PCR、Western blot检测也显示其成功表达目的蛋白，同时FCM分析显示CCR+CAR T细胞表面CCR+CAR的表达率为42%左右；ELISA检测CCR+CAR与表达GPC3和EPCAM的HepG2、Huh-7共培，其分泌更高水平的IL-2、INF-γ、IL-4,同时与人正常肝细胞L-02共培未发现CCR+CAR T细胞的有效激活。结论：成功获得组合型CCR+CAR T细胞，并初步探究其在肝癌细胞中的免疫效应，为后续动物体内免疫及实体瘤过继免疫治疗中防脱靶效应奠定基础。
Keyword ：
上皮细胞黏附分子 上皮细胞黏附分子 嵌合抗原共刺激受体 嵌合抗原共刺激受体 嵌合抗原受体 嵌合抗原受体 磷脂酰肌醇蛋白聚糖3 磷脂酰肌醇蛋白聚糖3
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| GB/T 7714 | 林海英 , 姚玢妍 , 于艺洁 et al. 组合型嵌合抗原受体的构建及分析鉴定 [J]. | 中国生物工程杂志 , 2023 , 43 (06) : 12-19 . |
| MLA | 林海英 et al. "组合型嵌合抗原受体的构建及分析鉴定" . | 中国生物工程杂志 43 . 06 (2023) : 12-19 . |
| APA | 林海英 , 姚玢妍 , 于艺洁 , 杨扬 . 组合型嵌合抗原受体的构建及分析鉴定 . | 中国生物工程杂志 , 2023 , 43 (06) , 12-19 . |
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To construct T cells encoding chimeric antigen receptor (CAR) targeting glypican-3 (GPC3) and chimeric antigen costimulatory receptor (CCR) targeting epithelial cell adhesion molecule (EPCAM), and analyze their activities in vitro. Methods: Gene fragments encoding EPCAM-CCR and GPC3- CAR was cloned into lentivirus vector plasmid. The recombinant CCR + CAR PCDH vector was identified by enzyme digestion, PCR and sequencing. T lymphocytes were isolated, activated and expanded. A stable combinatorial CAR modified cell line was generated using the lentivirus. Western blot, RT-PCR and flow cytometry (FCM) were used to verify the expression of CCR+ CAR in CCR+ CAR T cells. The secretion of cytokines IL-2, IL-4 and INF-y was detected by ELISA. Results: The CCR + CAR PCDH lentiviral recombinant plasmid was successfully constructed. Human T lymphocytes were successfully isolated, activated and expanded. Human T lymphocytes were successfully infected with CCR + CAR PCDH lentivirus. RT-PCR and Western blot showed that the target protein was successfully expressed, and FCM analysis showed that the expression rate of CCR + CAR on CCR + CAR T cells was about 42%. ELISA was used to detect CCR + CAR co-culture with HepG2 and Lu-7 expressing GPC3 and EPCAM, the secretion levels of IL-2, INF-y and IL-4 were higher, and the co-culture of human normal liver cells L-02 did not find effective activation of CCR+ CAR T cells. Conclusions: The combined CCR + CAR T cells were successfully obtained, and the immune effect of CAR T combined with the two antigen recognition signals was attempted in hepatocellular carcinoma cells. The result laid a foundation for the immune effects in the subsequent in vitro experiments, and provided a potentially novel approach to augment the off-target effect of CAR-T immunotherapy for solid tumors. © 2023, China Biotechnology Press. All rights reserved.
Keyword ：
Chimeric antigen costimulatory receptor Chimeric antigen costimulatory receptor Chimeric antigen receptor (CAR) Chimeric antigen receptor (CAR) Epithelial cell adhesion molecule Epithelial cell adhesion molecule Glypican-3 Glypican-3
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| GB/T 7714 | Lin, H.-Y. , Yao, B.-Y. , Yu, Y.-J. et al. Construction, Analysis and Characterization of Combinvssatorial Chimeric Antigen Receptor [J]. | China Biotechnology , 2023 , 43 (6) : 12-19 . |
| MLA | Lin, H.-Y. et al. "Construction, Analysis and Characterization of Combinvssatorial Chimeric Antigen Receptor" . | China Biotechnology 43 . 6 (2023) : 12-19 . |
| APA | Lin, H.-Y. , Yao, B.-Y. , Yu, Y.-J. , Yang, Y. . Construction, Analysis and Characterization of Combinvssatorial Chimeric Antigen Receptor . | China Biotechnology , 2023 , 43 (6) , 12-19 . |
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Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase IV in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells. ©2022 Chin J Biotech, All rights reserved.
Keyword ：
antioxidant enzyme antioxidant enzyme cell permeable cell permeable expression in Escherichia coli expression in Escherichia coli MMP-2/9 sensitive MMP-2/9 sensitive purification purification stability stability
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| GB/T 7714 | He, H. , Lin, L. , Li, L. et al. Expression, purification, and characterization of cell-permeable fusion antioxidant enzyme sensitive to matrix metalloproteinases-2/9 [基质金属蛋白酶-2/9 敏感型可跨膜融合抗氧化酶的表达、纯化和表征] [J]. | Chinese Journal of Biotechnology , 2022 , 38 (9) : 3515-3527 . |
| MLA | He, H. et al. "Expression, purification, and characterization of cell-permeable fusion antioxidant enzyme sensitive to matrix metalloproteinases-2/9 [基质金属蛋白酶-2/9 敏感型可跨膜融合抗氧化酶的表达、纯化和表征]" . | Chinese Journal of Biotechnology 38 . 9 (2022) : 3515-3527 . |
| APA | He, H. , Lin, L. , Li, L. , Wu, L. , Lin, H. , Pan, J. . Expression, purification, and characterization of cell-permeable fusion antioxidant enzyme sensitive to matrix metalloproteinases-2/9 [基质金属蛋白酶-2/9 敏感型可跨膜融合抗氧化酶的表达、纯化和表征] . | Chinese Journal of Biotechnology , 2022 , 38 (9) , 3515-3527 . |
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Abstract ：
融合了跨膜肽的抗氧化酶可进入细胞，保护细胞免受放射损伤。然而跨膜肽的跨膜能力没有靶向性，其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞，降低放疗的效果。为此，根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点，在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合MMP-2/9的底物肽X，设计了融合蛋白GST-SOD1-X-R9。该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨...
Keyword ：
MMP-2/9敏感性 MMP-2/9敏感性 可跨膜 可跨膜 大肠杆菌表达体系 大肠杆菌表达体系 抗氧化酶 抗氧化酶 稳定性 稳定性 纯化 纯化
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| GB/T 7714 | 何火聪 , 林丽香 , 李玲玲 et al. 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征（英文） [J]. | 生物工程学报 , 2022 , 38 (09) : 3515-3527 . |
| MLA | 何火聪 et al. "基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征（英文）" . | 生物工程学报 38 . 09 (2022) : 3515-3527 . |
| APA | 何火聪 , 林丽香 , 李玲玲 , 吴伦巧 , 林海英 , 潘剑茹 . 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征（英文） . | 生物工程学报 , 2022 , 38 (09) , 3515-3527 . |
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融合了跨膜肽的抗氧化酶可进入细胞,保护细胞免受放射损伤.然而跨膜肽的跨膜能力没有靶向性,其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞,降低放疗的效果.为此,根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点,在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合 MMP-2/9的底物肽X,设计了融合蛋白GST-SOD1-X-R9.该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨膜肽,从而无法进入肿瘤细胞,进而只能进入正常细胞.全基因合成SOD1-X-R9序列,并将其插入原核表达载体pGEX-4T-1中,得到表达质粒,并实现了GST-SOD1-X-R9融合蛋白的可溶表达.GST-SOD1-X-R9经硫酸铵沉淀和GST亲和层析纯化,分子量约为47kDa,与理论值一致.纯化的融合蛋白的SOD活性和GST活性分别为2 954 U/mg和328 U/mgoGST-SOD1-X-R9的SOD活性或GST活性在生理条件下几乎没有变化.该融合蛋白在溶液中可被胶原酶Ⅳ部分水解.分别建立了2D和3D培养的HepG2细胞模型来检验肿瘤微环境中的MMP-2活力对该蛋白跨膜能力的影响.在2D培养模型中,HepG2的MMP-2活力极低,但在3D培养模型中,随着培养时间的增加,HepG2肿瘤球的体积变大,其胞外MMP-2活力也随之增强.GST-SOD1-X-R9在2D培养的HepG2细胞中具有和GST-SOD1-R9蛋白一样的跨膜效率,但在3D培养的HepG2细胞球中的跨膜能力大大降低.本研究为后续深入研究GST-SOD1-X-R9靶向防护正常细胞的氧化损伤效应奠定了基础.
Keyword ：
MMP-2/9敏感性 MMP-2/9敏感性 可跨膜 可跨膜 大肠杆菌表达体系 大肠杆菌表达体系 抗氧化酶 抗氧化酶 稳定性 稳定性 纯化 纯化
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| GB/T 7714 | 何火聪 , 林丽香 , 李玲玲 et al. 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征 [J]. | 生物工程学报 , 2022 , 38 (9) : 3515-3527 . |
| MLA | 何火聪 et al. "基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征" . | 生物工程学报 38 . 9 (2022) : 3515-3527 . |
| APA | 何火聪 , 林丽香 , 李玲玲 , 吴伦巧 , 林海英 , 潘剑茹 . 基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征 . | 生物工程学报 , 2022 , 38 (9) , 3515-3527 . |
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The trans-translation system is recognized as an excellent target for developing new drugs to rapidly sterilize Mycobacterium tuberculosis (TB) infection and significantly shorten TB treatment duration. As a vital component of the trans-translation system for rescuing stalled ribosomes, the SmpB protein from Mycobacterium tuberculosis (MtbSmpB, 1-160 a. a.) mediates tmRNA binding to stalled ribosomes through forming a complex with tmRNA. So far, few works have been conducted to prepare, characterize biophysical properties and determine three-dimensional structure for the full-length MtbSmpB protein. In the present work, we successfully expressed and purified the His-tagged full-length MtbSmpB protein in Escherichia coli with a yield of 26.9 mg from 1 L of Luria Bertani medium. We also obtained MtbSmpB with a yield of 18.5 mg from 1 L of M9 minimal medium. The MtbSmpB protein showed a single band in SDS-PAGE with a molecular weight of similar to 20 kDa consistent with the measurement from MALDI-TOF-mass spectrometry. The dynamic light scattering experiment indicated that MtbSmpB existed in a monomeric form. Moreover, both circular dichroism and nuclear magnetic resonance (NMR) experiments exhibited that MtbSmpB was well structured, suggesting that it could be feasible to determine its solution structure by NMR spectroscopy. NMR titration experiments showed that MtbSmpB specifically bound to tmRNA. This work lays the essential basis for further determining the solution structure and dynamics of the full-length MtbSmpB protein.
Keyword ：
MtbSmpB MtbSmpB NMR NMR Protein expression and purification Protein expression and purification Protein-tmRNA interaction Protein-tmRNA interaction Tuberculosis Tuberculosis
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| GB/T 7714 | Yang, Juanjuan , Liu, Yindi , Xu, Shuli et al. Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis [J]. | PROTEIN EXPRESSION AND PURIFICATION , 2018 , 151 : 9-17 . |
| MLA | Yang, Juanjuan et al. "Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis" . | PROTEIN EXPRESSION AND PURIFICATION 151 (2018) : 9-17 . |
| APA | Yang, Juanjuan , Liu, Yindi , Xu, Shuli , Lin, Haiying , Meng, Chun , Lin, Donghai . Expression, purification and characterization of the full-length SmpB protein from Mycobacterium tuberculosis . | PROTEIN EXPRESSION AND PURIFICATION , 2018 , 151 , 9-17 . |
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