结核分枝杆菌RmlC的表达及酶活性鉴定
|
摘要
结核病(Tuberculosis,TB)是一种古老的疾病,是严重危害人类健康的疾病,也是全球关注的公共卫生问题和社会问题。其致病菌结核分枝杆菌(Mycobacterium tuberculosis)对为数很少的治疗药物均已产生了不同程度的耐药性,导致结核病患者的死亡率显著增加。近40年来人们一直在强烈地盼望新药面市,然而却未能如愿。因此,全球急需对付结核杆菌的有效措施,即研发有效的抗结核新药。
药物研发的关键是确定药物作用的靶标。结核分枝杆菌具有独特的细胞壁结构及生物合成和降解过程。并且细胞壁是维系分枝杆菌自身完整,生长繁殖和感染宿主的重要结构基础,故可作为新药开发的靶标。细胞壁中二糖衔接分子(L-鼠李糖-D-N-乙酰葡糖胺)是一个重要的结构成分,其中鼠李糖残基的供体是dTDP-鼠李糖,而且人体中不存在鼠李糖,因此,参与dTDP-鼠李糖合成的dTDP-鼠李糖合成酶系(RmlA,RmlB,RmlC和RmlD)是重要的研发新的抗结核药的靶标,抑制RmlA,RmlB,RmlC或RmlD酶的抑制剂,均可能成为新的抗结核药。RmlC酶,即dTDP-4-酮基-6-脱氧-D-葡萄糖3,5表异构酶,由rmlC基因所编码。最近的研究结果表明,rmlC基因是分枝杆菌生长必需基因。
为了建立体外筛选RmlC酶抑制剂的分子模型,我们需要获得高纯度的RmlC酶蛋白,并为建立快速、精确的酶活性测定方法和研究酶促反应动力学特性提供物质保障。我们实验室已克隆了rmlC基因并构建了在大肠杆菌中高表达rmlC基因的表达载体,在本研究中,我们将优化rmlC基因在大肠杆菌中的表
Tuberculosis (TB) is a kind of ancient disease, endangering the human being's health seriously, is also the public hygiene problem and social problems of the global concern. Major problem is the impressive emergence and wide distribution of multidrug-resistant strains of Mycobacterium tuberculosis. The mortality resulting from infection of M. tuberculosis has been increasing globally, but no tuberculosis-specific drugs have been discovered in 40 years. Therefore, there is a desperate need for new effective anti-TB drugs to prevent and treat TB.
The targets of new drugs need to be validated during drug discovery. The mycobacterial cell wall is required for the survival and growth of mycobacteria in the host, and has the unique components and structures. So, mycobacterial cell wall is a target to develop new anti-TB drugs. The disaccharidelinker (L-rhamnosyl-N-acetyl-glucosaminyl-phosphate) is fundamental to the structural integrity of the mycobacterial cell wall. The L-rhamnosyl residue in the disaccharide linker is provided with a sugar donor, dTDP-rhamnose (dTDP-Rha), and L-Rha is not found in humans. Therefore, the enzymes (RmlA, RmlB, RmlC and RmlD) involved in the formation of dTDP-Rha are important drug targets to develop new effective anti-TB drugs. The inhibitors that inhibit RmlA,RmlB,RmlC or RmlD could be new anti-TB drugs.
dTDP-4-keto-6-deoxy-D-glucose 3, 5 epimerase (RmlC) is encoded by rmlC gene. Recent research showed that rmlC is an essential gene for the survival and growth of mycobacteria.
In order to establish a molecular model based on RmlC enzyme assay to screen inhibitors of RmlC, a large amount of RmlC protein must be acquired. Soluble RmlC protein will be utilized for further development of a quick and accurate enzyme assay and study of
药物研发的关键是确定药物作用的靶标。结核分枝杆菌具有独特的细胞壁结构及生物合成和降解过程。并且细胞壁是维系分枝杆菌自身完整,生长繁殖和感染宿主的重要结构基础,故可作为新药开发的靶标。细胞壁中二糖衔接分子(L-鼠李糖-D-N-乙酰葡糖胺)是一个重要的结构成分,其中鼠李糖残基的供体是dTDP-鼠李糖,而且人体中不存在鼠李糖,因此,参与dTDP-鼠李糖合成的dTDP-鼠李糖合成酶系(RmlA,RmlB,RmlC和RmlD)是重要的研发新的抗结核药的靶标,抑制RmlA,RmlB,RmlC或RmlD酶的抑制剂,均可能成为新的抗结核药。RmlC酶,即dTDP-4-酮基-6-脱氧-D-葡萄糖3,5表异构酶,由rmlC基因所编码。最近的研究结果表明,rmlC基因是分枝杆菌生长必需基因。
为了建立体外筛选RmlC酶抑制剂的分子模型,我们需要获得高纯度的RmlC酶蛋白,并为建立快速、精确的酶活性测定方法和研究酶促反应动力学特性提供物质保障。我们实验室已克隆了rmlC基因并构建了在大肠杆菌中高表达rmlC基因的表达载体,在本研究中,我们将优化rmlC基因在大肠杆菌中的表
Tuberculosis (TB) is a kind of ancient disease, endangering the human being's health seriously, is also the public hygiene problem and social problems of the global concern. Major problem is the impressive emergence and wide distribution of multidrug-resistant strains of Mycobacterium tuberculosis. The mortality resulting from infection of M. tuberculosis has been increasing globally, but no tuberculosis-specific drugs have been discovered in 40 years. Therefore, there is a desperate need for new effective anti-TB drugs to prevent and treat TB.
The targets of new drugs need to be validated during drug discovery. The mycobacterial cell wall is required for the survival and growth of mycobacteria in the host, and has the unique components and structures. So, mycobacterial cell wall is a target to develop new anti-TB drugs. The disaccharidelinker (L-rhamnosyl-N-acetyl-glucosaminyl-phosphate) is fundamental to the structural integrity of the mycobacterial cell wall. The L-rhamnosyl residue in the disaccharide linker is provided with a sugar donor, dTDP-rhamnose (dTDP-Rha), and L-Rha is not found in humans. Therefore, the enzymes (RmlA, RmlB, RmlC and RmlD) involved in the formation of dTDP-Rha are important drug targets to develop new effective anti-TB drugs. The inhibitors that inhibit RmlA,RmlB,RmlC or RmlD could be new anti-TB drugs.
dTDP-4-keto-6-deoxy-D-glucose 3, 5 epimerase (RmlC) is encoded by rmlC gene. Recent research showed that rmlC is an essential gene for the survival and growth of mycobacteria.
In order to establish a molecular model based on RmlC enzyme assay to screen inhibitors of RmlC, a large amount of RmlC protein must be acquired. Soluble RmlC protein will be utilized for further development of a quick and accurate enzyme assay and study of
引文
1. World Health Organization. Global tuberculosis control: Surveillance, Planning, Financing. WHO Report 2005. Geneva, Switzerland, WHO/HTM/TB/2005.349.
2. O’Brien RJ. Tuberculosis. Scientific Blueprint for Tuberculosis Drug. Develop- ment. Tuberculosis (Edinb) 2001, 81(S1):1-52.
3. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Myobacterium tuberculosis from the complete genome sequence. Nature 1998, 393:537-544.
4. Dye C, Scheele S, Dolin P, et al. Global burden of tuberculosis estimated incidence, prevalence and mortality by country. JAMA 1999, 282(7):677-686.
5. Corbett EL, Watt CJ, Walker N, et al. The growing burden of tuberculosis: global trends and interaction with the HIV epidemic. Arch. Intern. Med. 2003, 163:1009-1021.
6. Murray CJL and Lopez AD. Global mortality, disability, and the contribution of risk factors:Global Burden of Disease Survey. Lancet 1997, 349(9063): 1436-1442.
7. O’Brien RJ. Tuberculosis. Scientific Blueprint for Tuberculosis Drug Development. Tuberculosis (Edinb) 2001, 81 (S1):1-52.
8. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393: 537-544.
9. Brennan PJ and Besra GS. Structure, function and biogenesis of the mycobacterial cell wall. Biochemical Society Transactions 1997, 25(1):188-94.
10. Khasnobis S, Escuyer VE and Chatterjee D. Emerging therapeutic targets in tuberculosis: post-genomic era. Expert Opinion on Therapeutic Targets 2002, 6(1):21-40.
11. Brennan PJ and Nikaido H. The envelope of mycobacteria. Annual Reviews of Biochemistry 1995, 64:29-63.
12. Crick DC, Brennan PJ and McNeil MR. The cell wall of Mycobacterium tuberculosis. In Tuberculosis (2 nd Edition), 2004, 115-134. Edited by Rom WN and Garay SM, Lippincott Williams & Wilkins, Philadelphia.
13. Ma Y, Mills JA, Belisle JT, et al. Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase. Microbiology 1997, 143:937-945.
14. Stern RJ, Lee TY, Lee TJ, et al. Conversion of dTDP-4-keto-6-Deoxyglucose to free dTDP-4-keto-Rhamnose by the rmlC gene products of Escherichia coli and Mycobacterium tuberculosis. Microbiology 1999, 145: 663-671.
15. Ma Y, Stern RJ, Scherman MS, et al. Drug targeting Mycobacterium tuberculosis cell wall synthesis: Genetics of dTDP-Rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-Rhamnose. Antimicrobial Agents and Chemotherapy 2001, 45(5):1407-1416.
16. Ma Y, Pan F and McNeil M. The formation of dTDP-rhamnose is essential for growth of mycobacteria. Journal of Bacteriology 2002, 184(12):3392-5.
17. Mills JA, Motichka K, Jucker M, et al. Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan- peptidoglycan linker, leads to irreversible loss of viability. Journal of Biological Chemistry 2004, 279(42):43540-43546.
18. Boles IC, Beerthuyzen MM, Kosters MHW, et al. Idenitification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-Rhamnose biosynthesis. Journal of Bacteriology 2004, 186(5):1239-1248.
19. Li W, Xin Y, McNeil MR, et al. rmlB and rmlC genes are essential for growth of mycobacteria. Biochemical and Biophysical Research Communications 2006, 342:170-178.
20. Sassetti CM, Boyd DH and Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Molecular Microbiology 2003, 48(1):77-84.
21. Bonner G, Lafer EM and Sousa R. Charaterizzation of a set of T7 RNA polymerase active site mutants. Journal of Biological Chemistry 1994, 269:25120-25128.
22. Chamberlin M, Mcgrath J and Waskell L. New RNA polymerase from ESchericha coli infected with bateriaphage T7. Nature 1970, 228:227-231.
23. Van Dyke MW, Sirito M and Sawadogo M. Single-step purification of bacterially expressed polypeptides containing an oligohistidine domain. Gene 1992, 111:99 -104.
24. Rosenberg AH, Lade BN, Chui DS, et al. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 1992, 56:125-135.
25. Studier FM and Moffatt BA. Use of bacteriophage T7 RNA polynerase to direct selective high-level expression of cloned gene. Journal of Molecular Biology1986, 189:113-130.
26. Studier FM, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to direct expression of clond gene. Methods of Enzymology 1990, 185:60-89.
27. Deng T, Noel JP and Tsai MD. A noval expression vetor for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine panareatic phospholipase A2. Gene 1990, 93:229-234.
28. Carrio MM and Villaverde A. Construction and deconstruction of bacterial inclusion bodies. Journal of Biotechnology 2002, 96(1):3-12.
29. Carrio MM, Cubarsi R and Villaverde A. Fine architecture of bacterial incusion bodies. FEBS Letter 2000, 471(1):7-11.
1. 蔡年生. 我国新药研究开发的进展与分析. 中国新药杂志, 1999, 8:73-74.
2. Koehn FE and Carter GT. The evolving role of natural products in drug discovery. Nature Reviews Drug discovery 2005, 4(3):206-220.
3. 杜冠华. 高通量药物筛选技术进展与新药开发策略. 中国新药杂志 2002, 11:31-36.
4. 于德泉. 从天然产物创制新药的趋势.中国工程科学 1999, 1(1):87-90.
5. 陈凯先, 蒋华良, 嵇汝运. 创新药物研究与生物技术. 中国科学院高技术发展报告. 北京出版社. 2001, pp98-106.
6. Boger DL, Fink BE and Hedrick MP. Total synthesis distamycin A and 2640 analogues:a solution-phase combinatorial approach to the discovery of new,bioactive DNA binding agents and development of a rapid,high-throughput screen for determining relative DNA binding affinity or DNA binding sequence selectivity. J Am Chem Sac 2000, 122-6382.
7. Liu Y and Sun Y. China traditional Chinese Medicine (TCM) Patent Database. World Patent Information 2004, 26:91 - 96.
8. Venter JC, Adams MD, Myera EW, et al . The Sequence of the Human Genome. Science 2001, 291 (5507): 304 -1351.
9. Lander E S, Linton L M, Birren B, et al. Initial sequencing and analysis of the human genome. Nature 2001, 409:860 - 921.
10. 秦伯益. 新药评价概论. 人民卫生出版社, 1998.
11. Searls DB. Using Bioinformatics in Gene Drug Discovery. Drug Discov Today. 2000, 5 (4):135-143.
12. May M and Heedneer G. Drug Discover and Biotechnologytrends. http:∥www. bioon.com/drug/ advance/chemdrug/2004/09/78814. html.
13. Moidor R, Sturn A, Michael M, et al. New trends in bioinformatics : fromgenme seguence to personalized medicine. Experimental Gerontology 2003, 38:1031-1036.
14. Anderson NL and Anderson NG. Proteome and proteomics: new techno logies, new concepts, and new words. Electrophoresis 1998, 19 (11):1853-1861.
15. Link AJ, Eng J, Schieltz DM, etal. Direct analysis of protein complexes using mass speclrometry. Techniques in microbial ecology 1999,17:676.
16. Gygi SP, Rist B, Gerber SA, et al. Quantitative analysis of complex proteinmixtures using isolope-code affinity tags. Techniques in microbial ecology 1999, 17:994.
17. Hu Y, Wang G and Grace YJ. Proteome analysis of saceharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis. Electrophoresis 2003, 24 (9):1488.
18. Debouck C and Metcalf B. The impact of genomicson drugs discovery. Annu Rev Pharmacol Toxicol 2000, 40:193-207.
19. TrainiM, Gooley AA, Wilkins MR, et al. Towards an automated approach for protein identification inproteome projects. Electrophoresis 1998, 19 (11):1941-1949.
20. WilkinsM R, Williams KL, Hohstrasser DF, et al. Proteome Research: New Frontiers in functional Genomics. New York: Springer, 1997.
21. Boguslavsky J. Biochip Market Explodes. Drug Discovery & Development 2001, 25.
22. Khan J, Bitter ML, Chen Y, et al. DNA microarray technology: the anticipated impact on the study of human disease. Biochem Biophys Acta, 1999, 1423:M17- 28.
23. Burbaum J. Miniaturization technologies in HTS: how fast, how small, how soon. Drug Discov Today 1998, 3:313-322.
24. Hensley P and Myszka DG. Analytical biotechnology Sorting needles and gaystacks. Curr Opin Biotech 2000, 11:9-12.
25. Sundberg SA. highthroughput and ultra- highthroughput screening:solution and cell-based approaches. Curr Opin Biotech, 2000, 11:47- 53.
26. Afshari CA, Nuwaysir EF and Barrett JC. Application of complementary DNA microarray technology to carcinogen identifications, toxicology, and drug safty evaluation. Cancer Res 1999, 59:4759-4760.
27. Debouck C. DNA microarrays in drug discovery and development. Nature Genetics Supplement 1999, 21:48.
28. Braxton S, Bedilion T. The integration of microarray information in the drug development process. Curr Opin Biotech 1998, 9:643.
29. Wachs T and Henion J. Electrospray device for coupling microscale separations and other miniaturizeddevices with electrospray mass spectrometry. Anal Chem 2001, 73(3):632 - 638.
30. Ramseier A, Heeren F and Thormann W. Analysis of fluorescein isothiocyanatederivatized amphetamine and analogs inhuman urine by capillary electrophoresis inchip-based and fused-silica-capillary instrumentation. Electrophoresis, 1998, 19 (16-17): 2967-2975.
31. Chiem N and Harrison DJ. Microchip-based capillary electrophoresis for immunoassays: analysis of monoclonal antibodies and theophylline. Anal Chem, 1997, 69 (3):373-378.
2. O’Brien RJ. Tuberculosis. Scientific Blueprint for Tuberculosis Drug. Develop- ment. Tuberculosis (Edinb) 2001, 81(S1):1-52.
3. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Myobacterium tuberculosis from the complete genome sequence. Nature 1998, 393:537-544.
4. Dye C, Scheele S, Dolin P, et al. Global burden of tuberculosis estimated incidence, prevalence and mortality by country. JAMA 1999, 282(7):677-686.
5. Corbett EL, Watt CJ, Walker N, et al. The growing burden of tuberculosis: global trends and interaction with the HIV epidemic. Arch. Intern. Med. 2003, 163:1009-1021.
6. Murray CJL and Lopez AD. Global mortality, disability, and the contribution of risk factors:Global Burden of Disease Survey. Lancet 1997, 349(9063): 1436-1442.
7. O’Brien RJ. Tuberculosis. Scientific Blueprint for Tuberculosis Drug Development. Tuberculosis (Edinb) 2001, 81 (S1):1-52.
8. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393: 537-544.
9. Brennan PJ and Besra GS. Structure, function and biogenesis of the mycobacterial cell wall. Biochemical Society Transactions 1997, 25(1):188-94.
10. Khasnobis S, Escuyer VE and Chatterjee D. Emerging therapeutic targets in tuberculosis: post-genomic era. Expert Opinion on Therapeutic Targets 2002, 6(1):21-40.
11. Brennan PJ and Nikaido H. The envelope of mycobacteria. Annual Reviews of Biochemistry 1995, 64:29-63.
12. Crick DC, Brennan PJ and McNeil MR. The cell wall of Mycobacterium tuberculosis. In Tuberculosis (2 nd Edition), 2004, 115-134. Edited by Rom WN and Garay SM, Lippincott Williams & Wilkins, Philadelphia.
13. Ma Y, Mills JA, Belisle JT, et al. Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase. Microbiology 1997, 143:937-945.
14. Stern RJ, Lee TY, Lee TJ, et al. Conversion of dTDP-4-keto-6-Deoxyglucose to free dTDP-4-keto-Rhamnose by the rmlC gene products of Escherichia coli and Mycobacterium tuberculosis. Microbiology 1999, 145: 663-671.
15. Ma Y, Stern RJ, Scherman MS, et al. Drug targeting Mycobacterium tuberculosis cell wall synthesis: Genetics of dTDP-Rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-Rhamnose. Antimicrobial Agents and Chemotherapy 2001, 45(5):1407-1416.
16. Ma Y, Pan F and McNeil M. The formation of dTDP-rhamnose is essential for growth of mycobacteria. Journal of Bacteriology 2002, 184(12):3392-5.
17. Mills JA, Motichka K, Jucker M, et al. Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan- peptidoglycan linker, leads to irreversible loss of viability. Journal of Biological Chemistry 2004, 279(42):43540-43546.
18. Boles IC, Beerthuyzen MM, Kosters MHW, et al. Idenitification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-Rhamnose biosynthesis. Journal of Bacteriology 2004, 186(5):1239-1248.
19. Li W, Xin Y, McNeil MR, et al. rmlB and rmlC genes are essential for growth of mycobacteria. Biochemical and Biophysical Research Communications 2006, 342:170-178.
20. Sassetti CM, Boyd DH and Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Molecular Microbiology 2003, 48(1):77-84.
21. Bonner G, Lafer EM and Sousa R. Charaterizzation of a set of T7 RNA polymerase active site mutants. Journal of Biological Chemistry 1994, 269:25120-25128.
22. Chamberlin M, Mcgrath J and Waskell L. New RNA polymerase from ESchericha coli infected with bateriaphage T7. Nature 1970, 228:227-231.
23. Van Dyke MW, Sirito M and Sawadogo M. Single-step purification of bacterially expressed polypeptides containing an oligohistidine domain. Gene 1992, 111:99 -104.
24. Rosenberg AH, Lade BN, Chui DS, et al. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 1992, 56:125-135.
25. Studier FM and Moffatt BA. Use of bacteriophage T7 RNA polynerase to direct selective high-level expression of cloned gene. Journal of Molecular Biology1986, 189:113-130.
26. Studier FM, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to direct expression of clond gene. Methods of Enzymology 1990, 185:60-89.
27. Deng T, Noel JP and Tsai MD. A noval expression vetor for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine panareatic phospholipase A2. Gene 1990, 93:229-234.
28. Carrio MM and Villaverde A. Construction and deconstruction of bacterial inclusion bodies. Journal of Biotechnology 2002, 96(1):3-12.
29. Carrio MM, Cubarsi R and Villaverde A. Fine architecture of bacterial incusion bodies. FEBS Letter 2000, 471(1):7-11.
1. 蔡年生. 我国新药研究开发的进展与分析. 中国新药杂志, 1999, 8:73-74.
2. Koehn FE and Carter GT. The evolving role of natural products in drug discovery. Nature Reviews Drug discovery 2005, 4(3):206-220.
3. 杜冠华. 高通量药物筛选技术进展与新药开发策略. 中国新药杂志 2002, 11:31-36.
4. 于德泉. 从天然产物创制新药的趋势.中国工程科学 1999, 1(1):87-90.
5. 陈凯先, 蒋华良, 嵇汝运. 创新药物研究与生物技术. 中国科学院高技术发展报告. 北京出版社. 2001, pp98-106.
6. Boger DL, Fink BE and Hedrick MP. Total synthesis distamycin A and 2640 analogues:a solution-phase combinatorial approach to the discovery of new,bioactive DNA binding agents and development of a rapid,high-throughput screen for determining relative DNA binding affinity or DNA binding sequence selectivity. J Am Chem Sac 2000, 122-6382.
7. Liu Y and Sun Y. China traditional Chinese Medicine (TCM) Patent Database. World Patent Information 2004, 26:91 - 96.
8. Venter JC, Adams MD, Myera EW, et al . The Sequence of the Human Genome. Science 2001, 291 (5507): 304 -1351.
9. Lander E S, Linton L M, Birren B, et al. Initial sequencing and analysis of the human genome. Nature 2001, 409:860 - 921.
10. 秦伯益. 新药评价概论. 人民卫生出版社, 1998.
11. Searls DB. Using Bioinformatics in Gene Drug Discovery. Drug Discov Today. 2000, 5 (4):135-143.
12. May M and Heedneer G. Drug Discover and Biotechnologytrends. http:∥www. bioon.com/drug/ advance/chemdrug/2004/09/78814. html.
13. Moidor R, Sturn A, Michael M, et al. New trends in bioinformatics : fromgenme seguence to personalized medicine. Experimental Gerontology 2003, 38:1031-1036.
14. Anderson NL and Anderson NG. Proteome and proteomics: new techno logies, new concepts, and new words. Electrophoresis 1998, 19 (11):1853-1861.
15. Link AJ, Eng J, Schieltz DM, etal. Direct analysis of protein complexes using mass speclrometry. Techniques in microbial ecology 1999,17:676.
16. Gygi SP, Rist B, Gerber SA, et al. Quantitative analysis of complex proteinmixtures using isolope-code affinity tags. Techniques in microbial ecology 1999, 17:994.
17. Hu Y, Wang G and Grace YJ. Proteome analysis of saceharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis. Electrophoresis 2003, 24 (9):1488.
18. Debouck C and Metcalf B. The impact of genomicson drugs discovery. Annu Rev Pharmacol Toxicol 2000, 40:193-207.
19. TrainiM, Gooley AA, Wilkins MR, et al. Towards an automated approach for protein identification inproteome projects. Electrophoresis 1998, 19 (11):1941-1949.
20. WilkinsM R, Williams KL, Hohstrasser DF, et al. Proteome Research: New Frontiers in functional Genomics. New York: Springer, 1997.
21. Boguslavsky J. Biochip Market Explodes. Drug Discovery & Development 2001, 25.
22. Khan J, Bitter ML, Chen Y, et al. DNA microarray technology: the anticipated impact on the study of human disease. Biochem Biophys Acta, 1999, 1423:M17- 28.
23. Burbaum J. Miniaturization technologies in HTS: how fast, how small, how soon. Drug Discov Today 1998, 3:313-322.
24. Hensley P and Myszka DG. Analytical biotechnology Sorting needles and gaystacks. Curr Opin Biotech 2000, 11:9-12.
25. Sundberg SA. highthroughput and ultra- highthroughput screening:solution and cell-based approaches. Curr Opin Biotech, 2000, 11:47- 53.
26. Afshari CA, Nuwaysir EF and Barrett JC. Application of complementary DNA microarray technology to carcinogen identifications, toxicology, and drug safty evaluation. Cancer Res 1999, 59:4759-4760.
27. Debouck C. DNA microarrays in drug discovery and development. Nature Genetics Supplement 1999, 21:48.
28. Braxton S, Bedilion T. The integration of microarray information in the drug development process. Curr Opin Biotech 1998, 9:643.
29. Wachs T and Henion J. Electrospray device for coupling microscale separations and other miniaturizeddevices with electrospray mass spectrometry. Anal Chem 2001, 73(3):632 - 638.
30. Ramseier A, Heeren F and Thormann W. Analysis of fluorescein isothiocyanatederivatized amphetamine and analogs inhuman urine by capillary electrophoresis inchip-based and fused-silica-capillary instrumentation. Electrophoresis, 1998, 19 (16-17): 2967-2975.
31. Chiem N and Harrison DJ. Microchip-based capillary electrophoresis for immunoassays: analysis of monoclonal antibodies and theophylline. Anal Chem, 1997, 69 (3):373-378.