7-磷酸有效氧胺合成酶ValL的结构研究
摘要
作为在亚洲广泛使用的农用抗生素和多种医用药物的前体,井冈霉素在农业及医学上具有非常重要的应用价值。因此,近年来井冈霉素的开发和研究是工业界和学术界倍受关注的问题。工业上发酵条件的优化,使得其每升产量增加了9倍,大大降低了井冈霉素的价格。同时,井冈霉素生物合成途径的研究证明了合成所必需的关键酶的存在,但是这些酶的结构和催化机理至今尚不明确。
本课题首先运用基因克隆技术将GST片段及TEV酶切位点片段重组入pET-28a(+)载体以构建新型原核表达载体pET-28a-GST-TEV,再将目的片段valL克隆到上述载体并于大肠杆菌BL21(DE3) Rosetta中进行蛋白表达,随后将表达的蛋白用于结晶,并解析晶体结构。
按照上述技术路线,本课题成功得到了带有His标签和GST标签的新型原核表达载体pET-28a-GST-TEV,大大提高了真核蛋白异源表达的可溶性,并能有效帮助蛋白折叠,具有较高的推广使用价值。同时本课题获得了高质量的ValL及其硒代衍生物晶体结构数据,其中衍射分辨率分别为2.0?和1.7?,成功解析出了ValL及ValL/海藻糖复合物的结构模型。结果表明ValL的整体结构与经典的“GT-B”葡萄糖转移酶非常相似,它含有两个结构域,每个结构域含有一个β/α/βRossman折叠,其活性中心位于两结构域的交界处。同时发现在ValL结构C端区域的最后两个螺旋间有一个拐点,使得螺旋上的氨基酸残基与N端区域相互作用,这也是GT-B酶家族的共同特点。此外,N端和C端交界处的电子云密度图很清楚地显示了ValL产物类似物海藻糖的位置。
本课题阐明了井冈霉素生物合成8个必需基因之一valL编码的7-磷酸有效氧胺合成酶的结构,为进一步了解井冈霉素的合成机理奠定了基础,同时也为在农业、医学等相关方面深度利用井冈霉素提供了理论切入点。
As a widely-used agricultural antibiotic in Asia and a precursor of various medicines, validamycin A bears extraordinary value for agriculture as well as pharmaceutical industry. Therefore, in recent years, the research and development of validamycin A has drawn significant attention from academia and industry alike. The optimization of fermentation condition has lead to ten-fold increase in yield per liter, which caused large drop in the price of validamycin A. Meanwhile, the study on its biosynthesis pathway has demonstrated the essential enzymes required for the validamycin A production. However, the structures and mechanisms of these enzymes on the pathway remain unclear. In this study, in order to construct the new prokaryotic expression vector pET-28a-GST-TEV we cloned the gene of GST and the cleavage site of TEV protease into pET-28a(+) plasmid. Then the target gene valL was inserted into pET 28a-GST-TEV and the recombinant protein was expressed in BL21(DE3) Rosetta cells. Subsequently, the protein was used to grow crystals and x-rays diffraction was chosen as the method to determine the three-dimensional structure.
Using the protocol described above, we construct a novel prokaryotic expression vector pET-28a-GST-TEV featuring His tag and GST Tag. It would have good application in the hetero expression of eukaryotic genes. In addition, we collected the diffraction data of native ValL crystals as well as selenomethionine-labelled ValL crystals with resolutions of 1.9? and 1.7? respectively. Based on the above data, we built the three dimensional models of ValL and ValL/trehalose complex. The structural analysis shows that the overall structure of ValL closely resembles the classic fold of‘‘GT-B’’glycosyltransferases. It harbors two domains each of which contains aβ/α/βRossmann fold and an active site at the interface of them. Also found in the ValL structure is a kink between last two C-terminal helices, which is the common feature of GT-B enzyme family. The kink directs the last C-terminal helix into the vicinity of N-terminal domain and causes the residues in the helix to interact with the N-terminal domain. Furthermore, the electron density at the junction of N-terminal and C-terminal unambiguously indicates the location of trehalose embedded in the active site.
In this work, we solved the structure of validoxylamine 7-phosphate synthase encoded by one of the eight essential genes required for validamycin biosynthesis. The results laid the framework for the further study of synthetic mechanism of validamycin and they are instrumental for the advanced application of validamycin in agriculture as well as medical science.
本课题首先运用基因克隆技术将GST片段及TEV酶切位点片段重组入pET-28a(+)载体以构建新型原核表达载体pET-28a-GST-TEV,再将目的片段valL克隆到上述载体并于大肠杆菌BL21(DE3) Rosetta中进行蛋白表达,随后将表达的蛋白用于结晶,并解析晶体结构。
按照上述技术路线,本课题成功得到了带有His标签和GST标签的新型原核表达载体pET-28a-GST-TEV,大大提高了真核蛋白异源表达的可溶性,并能有效帮助蛋白折叠,具有较高的推广使用价值。同时本课题获得了高质量的ValL及其硒代衍生物晶体结构数据,其中衍射分辨率分别为2.0?和1.7?,成功解析出了ValL及ValL/海藻糖复合物的结构模型。结果表明ValL的整体结构与经典的“GT-B”葡萄糖转移酶非常相似,它含有两个结构域,每个结构域含有一个β/α/βRossman折叠,其活性中心位于两结构域的交界处。同时发现在ValL结构C端区域的最后两个螺旋间有一个拐点,使得螺旋上的氨基酸残基与N端区域相互作用,这也是GT-B酶家族的共同特点。此外,N端和C端交界处的电子云密度图很清楚地显示了ValL产物类似物海藻糖的位置。
本课题阐明了井冈霉素生物合成8个必需基因之一valL编码的7-磷酸有效氧胺合成酶的结构,为进一步了解井冈霉素的合成机理奠定了基础,同时也为在农业、医学等相关方面深度利用井冈霉素提供了理论切入点。
As a widely-used agricultural antibiotic in Asia and a precursor of various medicines, validamycin A bears extraordinary value for agriculture as well as pharmaceutical industry. Therefore, in recent years, the research and development of validamycin A has drawn significant attention from academia and industry alike. The optimization of fermentation condition has lead to ten-fold increase in yield per liter, which caused large drop in the price of validamycin A. Meanwhile, the study on its biosynthesis pathway has demonstrated the essential enzymes required for the validamycin A production. However, the structures and mechanisms of these enzymes on the pathway remain unclear. In this study, in order to construct the new prokaryotic expression vector pET-28a-GST-TEV we cloned the gene of GST and the cleavage site of TEV protease into pET-28a(+) plasmid. Then the target gene valL was inserted into pET 28a-GST-TEV and the recombinant protein was expressed in BL21(DE3) Rosetta cells. Subsequently, the protein was used to grow crystals and x-rays diffraction was chosen as the method to determine the three-dimensional structure.
Using the protocol described above, we construct a novel prokaryotic expression vector pET-28a-GST-TEV featuring His tag and GST Tag. It would have good application in the hetero expression of eukaryotic genes. In addition, we collected the diffraction data of native ValL crystals as well as selenomethionine-labelled ValL crystals with resolutions of 1.9? and 1.7? respectively. Based on the above data, we built the three dimensional models of ValL and ValL/trehalose complex. The structural analysis shows that the overall structure of ValL closely resembles the classic fold of‘‘GT-B’’glycosyltransferases. It harbors two domains each of which contains aβ/α/βRossmann fold and an active site at the interface of them. Also found in the ValL structure is a kink between last two C-terminal helices, which is the common feature of GT-B enzyme family. The kink directs the last C-terminal helix into the vicinity of N-terminal domain and causes the residues in the helix to interact with the N-terminal domain. Furthermore, the electron density at the junction of N-terminal and C-terminal unambiguously indicates the location of trehalose embedded in the active site.
In this work, we solved the structure of validoxylamine 7-phosphate synthase encoded by one of the eight essential genes required for validamycin biosynthesis. The results laid the framework for the further study of synthetic mechanism of validamycin and they are instrumental for the advanced application of validamycin in agriculture as well as medical science.
引文
[1]刘海民.井冈霉素防治水稻纹枯病作用机理研究[D].福建:福建农林大学, 2008
[2]李顺德,黄兰英.井冈霉素防治玉米纹枯病的一种施药新技术[J].农药, 1999, 38(1): 35
[3]陈小龙,方夏,沈寅初.纹枯病菌对井冈霉素的作用机制、抗药性及安全性[J].农药, 2010, 49(7): 481-483
[4]白林泉,章亦荣,徐慧,等.井冈霉素生物合成的基础研究及其应用潜力[C].见:中国微生物学会《第二届全国农业微生物研究及产业研讨会》和《第十一届全国杀虫微生物学术研讨会》暨《湖北省暨武汉市微生物学会和内蒙古微生物学会2008年会》论文集.中国内蒙古呼和浩特: 2008: 39-40
[5]郭文灿,钟世华,黄叶菊,等.井冈霉素生产工艺优化研究[J]. 2006, 36(4): 40-42
[6]徐慧.井冈霉素的生物合成及途径改造[D].上海:上海交通大学, 2009
[7]黄昭穗,周丽芳,黄胜立,等.伏格列波糖对II型糖尿病作用的临床观察[J].中国医院药学杂志, 2000, 20(9): 554-556
[8]赵学明.代谢物组学与工业微生物代谢工程[C].见:代谢工程和工业微生物技术研讨会讨论文集.中国山东日照: 2004: 3
[9] Sun X, Duan Y R, He Q, et al. PELGE nanoparticles as new carriers for the delivery of plasmid DNA[J]. Chem Pharm Bull (Tokyo), 2005, 53(6): 599-603
[10]陈俊升,景兰,邵雷.组合生物合成技术在药物研发中的应用[J].药学进展, 2009, 33(8): 337-343
[11] Asano N, Kameda Y, Matsui K, et al. Validamycin H, a new pseudo-tetrasaccharide antibiotic[J]. J Antibiot (Tokyo), 1990, 43(8): 1039-1041
[12]申屠旭萍,俞晓平,吕仲贤,等.海藻糖酶抑制剂—井冈霉亚基胺A的研究进展[C].见:当代昆虫学研究——中国昆虫学会成立60周年纪念大会暨学术讨论会论文集.北京: 2004: 261-264
[13] Asano N, Yamaguchi T, Kameda Y, et al. Effect of validamycins on glycohydrolases of Rhizoctonia solani[J]. J Antibiot (Tokyo), 1987, 40(4): 526-532
[14]董华平,郑裕国.井冈霉亚基胺A C-N键裂解酶的分离纯化[C].见:第一届全国化学工程与生物化工年会论文集.南京: 2004: 876
[15]王春辉.井冈胺产生菌的选育与工艺条件的研究[D].浙江:浙江工业大学, 2004
[16]李磊,白林泉,邓子新.井冈霉亚基胺A高产突变株的构建[J].上海交通大学学报, 2008, 42(5): 689-692
[17] Degwert U, Pape H, Beale J M, et al. Studies on the biosynthesis of the alpha-glucosidase inhibitor acarbose: valienamine, a m-C7N unit not derived from the shikimate pathway[J]. J Antibiot (Tokyo), 1987, 40(6): 855-861
[18] Jin, W Z, Rinehart K J, Toyokuni T. Biosynthetic studies on validamycins. I. 1H and 13C NMR assignments of validamycin A[J]. J Antibiot (Tokyo), 1987, 40(3): 329-339
[19] Dong H, Mahmud T, Tornus I, et al. Biosynthesis of the validamycins: identification of intermediates in the biosynthesis of validamycin A by Streptomyces hygroscopicus var. limoneus[J]. J Am Chem Soc, 2001, 123(12): 2733-2742
[20] Yu Y, Bai L, Minagawa K, et al. Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008[J]. Appl Environ Microbiol, 2005, 71(9): 5066-5076
[21]章亦荣.井冈霉素与杀念菌素生物合成基因的功能研究[D].上海:上海交通大学, 2009
[22] Minagawa K, Zhang Y, Ito T, et al. ValC, a new type of C7-Cyclitol kinase involved in the biosynthesis of the antifungal agent validamycin A[J]. Chembiochem, 2007, 8(6): 632-641
[23]徐慧,白林泉,邓子新.井冈霉素生物合成基因的重组装与异源表达[C].见: 2006中国微生物学会第九次全国会员代表大会暨学术年会论文集.中国湖北武汉: 2006: 115
[24] Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans[J]. Plant Cell, 1990, 2(4): 279-289
[25] Burley S K, Almo S C, Bonanno J B, et al. Structural genomics: beyond the human genome project[J]. Nat Genet, 1999, 23(2): 151-157
[26] Sali A. 100,000 protein structures for the biologist[J]. Nat Struct Biol, 1998, 5(12): 1029-1032
[27] Burley S K. An overview of structural genomics[J]. Nat Struct Biol, 2000, 7: 932-934
[28] Kim S H. Shining a light on structural genomics[J]. Nat Struct Biol, 1998, 5: 643-5
[29] Watts A, Straus S K, Grage S L, et al. Membrane protein structure determination using solid-state NMR[J]. Methods Mol Biol, 2004, 278: 403-473
[30] Hansen G, Hercus T R, Xu Y, et al. Crystallization and preliminary X-ray diffraction analysis of the ternary human GM-CSF receptor complex[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2008, 64(8): 711-714
[31] Chayen N E, Saridakis E. Protein crystallization: from purified protein to diffraction-quality crystal[J]. Nat Methods, 2008, 5(2): 147-153
[32] Helliwell J R, Chayen N E. Crystallography: a down-to-Earth approach[J]. Nature, 2007, 448(7154): 658-659
[33] Chayen N E. Comparative studies of protein crystallization by vapour-diffusion and microbatch techniques[J]. Acta Crystallogr D Biol Crystallogr, 1998, D 54(1): 8-15
[34]鹿芹芹,尹大川,刘永明,等.提高蛋白质晶体质量的研究进展[J].材料导报, 2010, 24(1): 104-110
[35] Lee T S, Vaghjiani J D, Lye G J, et al. A systematic approach to the large-scale production of protein crystals[J]. Enzyme Microb Technol, 2000, 26(8): 582-592
[36] Klyushnichenko V. Protein crystallization: from HTS to kilogram-scale[J]. CurrOpin Drug Discov Devel, 2003, 6(6): 848-854
[37] Anderson M J, Hansen C L, Quake S R. Phase knowledge enables rational screens for protein crystallization[J]. Proc Natl Acad Sci U S A, 2006, 103(45): 16746-16751
[38] McPherson A. Introduction to protein crystallization[J]. Methods, 2004, 34(3): 254-265
[39] Rupp B, Wang J. Predictive models for protein crystallization[J]. Methods, 2004, 34(3): 390-407
[40] Heijna M C R, van Enckevort W J P, Vlieg E. Crystal growth in a three-phase system: diffusion and liquid-liquid phase separation in lysozyme crystal growth[J]. Phys Rev E Stat Nonlin Soft Matter Phys, 2007, 76(1): 011604-011614
[41] Adachi H, Takano K, Matsumusa H, et al. Protein crystal growth with a two-liquid system and stirring solution[J]. J Synchrotron Radiat, 2004, 11(1): 121-124
[42] Rupp B, Wang J. Predictive models for protein crystallization[J]. Methods, 2004, 34(3): 390-407
[43]陶凤云,张新妙,马润宇.蛋白质结晶过程中的影响因素[J].化学工业与工程, 2006, 23(3): 260-264
[44]王弘,于淑娟,高大维.蛋白质大分子的结晶[J].生命的化学, 2001, 21(5): 429-431
[45]戴国亮,于泳,康琦,等.溶菌酶晶体生长前期溶液中聚集体研究[J].化学学报, 2004, 62(8): 757-761
[46] Majeed S, Ofek G, Belachew A, et al. Enhancing protein crystallization through precipitant synergy[J]. Structure, 2003, 11(9): 1061-1070
[47]孙百虎,尚平.蛋白质晶体生长方法综述[J].石家庄职业技术学院报, 2010, 22(4): 12-15
[48] Zeppezauer M, Eklund H, Zeppezauer E S. Micro diffusion cells for the growth of single protein crystals by means of equilibrium dialysis[J]. Arch Biochem Biophys,1968, 126(2): 564-573
[49] Kundrot C E. Which strategy for a protein crystallization project[J]. Cell Mol Life Sci, 2004, 61(5): 525-36
[50] Moradian F, Garen C, Cherney L, et al. Expression, purification, crystallization and preliminary X-ray analysis of two arginine-biosynthetic enzymes from Mycobacterium tuberculosis[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2006, 62(10): 986-988
[51] Berman H M, Westbrook J D, Gabanyi M J, et al. The protein structure initiative structural genomics knowledgebase[J]. Nucleic Acids Res, 2009, 37(Database issue): D365-368
[52]李秀宏.上海光源在生命科学和高分子材料中的应用[J].现代物理知识, 2010, 22(3): 20-28
[53] Congreve M, Murray C W, Blundell T L. Structural biology and drug discovery[J]. Curr Pharm Des, 2005, 10(13): 895-907
[2]李顺德,黄兰英.井冈霉素防治玉米纹枯病的一种施药新技术[J].农药, 1999, 38(1): 35
[3]陈小龙,方夏,沈寅初.纹枯病菌对井冈霉素的作用机制、抗药性及安全性[J].农药, 2010, 49(7): 481-483
[4]白林泉,章亦荣,徐慧,等.井冈霉素生物合成的基础研究及其应用潜力[C].见:中国微生物学会《第二届全国农业微生物研究及产业研讨会》和《第十一届全国杀虫微生物学术研讨会》暨《湖北省暨武汉市微生物学会和内蒙古微生物学会2008年会》论文集.中国内蒙古呼和浩特: 2008: 39-40
[5]郭文灿,钟世华,黄叶菊,等.井冈霉素生产工艺优化研究[J]. 2006, 36(4): 40-42
[6]徐慧.井冈霉素的生物合成及途径改造[D].上海:上海交通大学, 2009
[7]黄昭穗,周丽芳,黄胜立,等.伏格列波糖对II型糖尿病作用的临床观察[J].中国医院药学杂志, 2000, 20(9): 554-556
[8]赵学明.代谢物组学与工业微生物代谢工程[C].见:代谢工程和工业微生物技术研讨会讨论文集.中国山东日照: 2004: 3
[9] Sun X, Duan Y R, He Q, et al. PELGE nanoparticles as new carriers for the delivery of plasmid DNA[J]. Chem Pharm Bull (Tokyo), 2005, 53(6): 599-603
[10]陈俊升,景兰,邵雷.组合生物合成技术在药物研发中的应用[J].药学进展, 2009, 33(8): 337-343
[11] Asano N, Kameda Y, Matsui K, et al. Validamycin H, a new pseudo-tetrasaccharide antibiotic[J]. J Antibiot (Tokyo), 1990, 43(8): 1039-1041
[12]申屠旭萍,俞晓平,吕仲贤,等.海藻糖酶抑制剂—井冈霉亚基胺A的研究进展[C].见:当代昆虫学研究——中国昆虫学会成立60周年纪念大会暨学术讨论会论文集.北京: 2004: 261-264
[13] Asano N, Yamaguchi T, Kameda Y, et al. Effect of validamycins on glycohydrolases of Rhizoctonia solani[J]. J Antibiot (Tokyo), 1987, 40(4): 526-532
[14]董华平,郑裕国.井冈霉亚基胺A C-N键裂解酶的分离纯化[C].见:第一届全国化学工程与生物化工年会论文集.南京: 2004: 876
[15]王春辉.井冈胺产生菌的选育与工艺条件的研究[D].浙江:浙江工业大学, 2004
[16]李磊,白林泉,邓子新.井冈霉亚基胺A高产突变株的构建[J].上海交通大学学报, 2008, 42(5): 689-692
[17] Degwert U, Pape H, Beale J M, et al. Studies on the biosynthesis of the alpha-glucosidase inhibitor acarbose: valienamine, a m-C7N unit not derived from the shikimate pathway[J]. J Antibiot (Tokyo), 1987, 40(6): 855-861
[18] Jin, W Z, Rinehart K J, Toyokuni T. Biosynthetic studies on validamycins. I. 1H and 13C NMR assignments of validamycin A[J]. J Antibiot (Tokyo), 1987, 40(3): 329-339
[19] Dong H, Mahmud T, Tornus I, et al. Biosynthesis of the validamycins: identification of intermediates in the biosynthesis of validamycin A by Streptomyces hygroscopicus var. limoneus[J]. J Am Chem Soc, 2001, 123(12): 2733-2742
[20] Yu Y, Bai L, Minagawa K, et al. Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008[J]. Appl Environ Microbiol, 2005, 71(9): 5066-5076
[21]章亦荣.井冈霉素与杀念菌素生物合成基因的功能研究[D].上海:上海交通大学, 2009
[22] Minagawa K, Zhang Y, Ito T, et al. ValC, a new type of C7-Cyclitol kinase involved in the biosynthesis of the antifungal agent validamycin A[J]. Chembiochem, 2007, 8(6): 632-641
[23]徐慧,白林泉,邓子新.井冈霉素生物合成基因的重组装与异源表达[C].见: 2006中国微生物学会第九次全国会员代表大会暨学术年会论文集.中国湖北武汉: 2006: 115
[24] Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans[J]. Plant Cell, 1990, 2(4): 279-289
[25] Burley S K, Almo S C, Bonanno J B, et al. Structural genomics: beyond the human genome project[J]. Nat Genet, 1999, 23(2): 151-157
[26] Sali A. 100,000 protein structures for the biologist[J]. Nat Struct Biol, 1998, 5(12): 1029-1032
[27] Burley S K. An overview of structural genomics[J]. Nat Struct Biol, 2000, 7: 932-934
[28] Kim S H. Shining a light on structural genomics[J]. Nat Struct Biol, 1998, 5: 643-5
[29] Watts A, Straus S K, Grage S L, et al. Membrane protein structure determination using solid-state NMR[J]. Methods Mol Biol, 2004, 278: 403-473
[30] Hansen G, Hercus T R, Xu Y, et al. Crystallization and preliminary X-ray diffraction analysis of the ternary human GM-CSF receptor complex[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2008, 64(8): 711-714
[31] Chayen N E, Saridakis E. Protein crystallization: from purified protein to diffraction-quality crystal[J]. Nat Methods, 2008, 5(2): 147-153
[32] Helliwell J R, Chayen N E. Crystallography: a down-to-Earth approach[J]. Nature, 2007, 448(7154): 658-659
[33] Chayen N E. Comparative studies of protein crystallization by vapour-diffusion and microbatch techniques[J]. Acta Crystallogr D Biol Crystallogr, 1998, D 54(1): 8-15
[34]鹿芹芹,尹大川,刘永明,等.提高蛋白质晶体质量的研究进展[J].材料导报, 2010, 24(1): 104-110
[35] Lee T S, Vaghjiani J D, Lye G J, et al. A systematic approach to the large-scale production of protein crystals[J]. Enzyme Microb Technol, 2000, 26(8): 582-592
[36] Klyushnichenko V. Protein crystallization: from HTS to kilogram-scale[J]. CurrOpin Drug Discov Devel, 2003, 6(6): 848-854
[37] Anderson M J, Hansen C L, Quake S R. Phase knowledge enables rational screens for protein crystallization[J]. Proc Natl Acad Sci U S A, 2006, 103(45): 16746-16751
[38] McPherson A. Introduction to protein crystallization[J]. Methods, 2004, 34(3): 254-265
[39] Rupp B, Wang J. Predictive models for protein crystallization[J]. Methods, 2004, 34(3): 390-407
[40] Heijna M C R, van Enckevort W J P, Vlieg E. Crystal growth in a three-phase system: diffusion and liquid-liquid phase separation in lysozyme crystal growth[J]. Phys Rev E Stat Nonlin Soft Matter Phys, 2007, 76(1): 011604-011614
[41] Adachi H, Takano K, Matsumusa H, et al. Protein crystal growth with a two-liquid system and stirring solution[J]. J Synchrotron Radiat, 2004, 11(1): 121-124
[42] Rupp B, Wang J. Predictive models for protein crystallization[J]. Methods, 2004, 34(3): 390-407
[43]陶凤云,张新妙,马润宇.蛋白质结晶过程中的影响因素[J].化学工业与工程, 2006, 23(3): 260-264
[44]王弘,于淑娟,高大维.蛋白质大分子的结晶[J].生命的化学, 2001, 21(5): 429-431
[45]戴国亮,于泳,康琦,等.溶菌酶晶体生长前期溶液中聚集体研究[J].化学学报, 2004, 62(8): 757-761
[46] Majeed S, Ofek G, Belachew A, et al. Enhancing protein crystallization through precipitant synergy[J]. Structure, 2003, 11(9): 1061-1070
[47]孙百虎,尚平.蛋白质晶体生长方法综述[J].石家庄职业技术学院报, 2010, 22(4): 12-15
[48] Zeppezauer M, Eklund H, Zeppezauer E S. Micro diffusion cells for the growth of single protein crystals by means of equilibrium dialysis[J]. Arch Biochem Biophys,1968, 126(2): 564-573
[49] Kundrot C E. Which strategy for a protein crystallization project[J]. Cell Mol Life Sci, 2004, 61(5): 525-36
[50] Moradian F, Garen C, Cherney L, et al. Expression, purification, crystallization and preliminary X-ray analysis of two arginine-biosynthetic enzymes from Mycobacterium tuberculosis[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2006, 62(10): 986-988
[51] Berman H M, Westbrook J D, Gabanyi M J, et al. The protein structure initiative structural genomics knowledgebase[J]. Nucleic Acids Res, 2009, 37(Database issue): D365-368
[52]李秀宏.上海光源在生命科学和高分子材料中的应用[J].现代物理知识, 2010, 22(3): 20-28
[53] Congreve M, Murray C W, Blundell T L. Structural biology and drug discovery[J]. Curr Pharm Des, 2005, 10(13): 895-907