N-乙酰-D-神经氨酸的酶法合成和正选择表达载体的构建
|
摘要
N-乙酰-D-神经氨酸是化学合成扎那米韦的关键中间体,也是合成其他抗流感药物的主要原料。目前,N-乙酰-D-神经氨酸主要通过酶催化法制备。首先由N-酰基-D-葡萄糖胺2-异构酶将N-乙酰-D-葡萄糖胺异构化为N-乙酰-D-甘露糖胺；从后者到N-乙酰-D-神经氨酸有两个途径：一种是神经氨酸醛缩酶催化N-乙酰-D-甘露糖胺与丙酮酸之间的缩合而得到N-乙酰-D-神经氨酸；另一种是神经氨酸合成酶催化N-乙酰-D-甘露糖胺与磷酸烯醇式丙酮酸缩合为N-乙酰-D-神经氨酸。
本研究通过同源搜索找到与已知神经氨酸合成酶具有较高同源性的蛋白,进而在大肠杆菌中进行克隆、表达和纯化。同时测定了不同来源的神经氨酸醛缩酶、神经氨酸合成酶以及N-酰基-D-葡萄糖胺2-异构酶的活性。结果表明来源于大肠杆菌的醛缩酶活性最高,以全细胞进行催化,37℃下反应1h后,神经氨酸的摩尔收率最高可以达到23.31%；来源于脑膜炎双球菌的神经氨酸合成酶在全细胞水平具有很高的合成酶活性,37℃下全细胞催化反应30分钟后Neu5Ac的摩尔收率最高可以达到50.96%；来源于多拟形杆菌的BT0453蛋白的异构酶活性较好,与大肠杆菌醛缩酶偶联反应,反应时间为4h后,神经氨酸的摩尔收率最高达到2.77%。
目前将来源自大肠杆菌的三种伴侣蛋白作为融合标签构建了伴侣蛋白融合表达载体。选取A113695和RB3348与伴侣蛋白基因融合表达。结果表明,目的基因与三种伴侣蛋白融合表达的重组蛋白可溶性明显增加,启动因子作为融合标签的融合蛋白表达量最高。BandScan软件分析表明,a113695基因与启动因子基因融合表达时,表达出95.5KD融合蛋白,所表达的融合蛋白几乎均为可溶性蛋白,其可溶性蛋白的表达量占总表达蛋白的比例为37.2%,高于a113695单独表达时的7.1%的比例；RB3348基因与启动因子基因融合表达时,可溶性蛋白的表达量占总表达蛋白的比例为53.0%,远高于单独表达时4.4%的比例。
正选择克隆载体是一类通过抗生素或化合物对重组克隆进行直接筛选的载体,最大优点是其可保证很高的克隆效率,有时可达100%。有报道显示β-内酰胺酶基因(bla)在缺失C端密码子(290位色氨酸密码子)时,将不能很好的表达氨苄青霉素抗性功能；而定向插入外源基因后将重建bla的C端密码子,进而使β-内酰胺酶重获氨苄青霉素抗性功能。本研究将色氨酸缺失的β-内酰胺酶基因克隆至带有TEV蛋白酶酶切位点的表达载体上,构建了正选择表达载体pPAE以及pPAE-MBP。选取大肠杆菌醛缩酶nanA和人源肾素结合蛋白hRnBP做克隆范例。实验结果证明pPAE和pPAE-MBP的克隆效率均达100%,同时所克隆的基因成功在大肠杆菌中表达。
N-Acetyl-D-neuraminic acid (Neu5Ac) is the key intermediate to chemical synthesis of Zanamivir, one of the two medicines used in the treatment of influenza. N-acetyl-D-neuraminic acid is currently made through enzymatic synthesis; the procedure consists of two steps:first, N-acetyl-D-glucosamine 2-epimerase (AGE) catalyzes the transformation of N-acetyl-D-glucosamine (GlcNAc) into N-acetyl-D-mannosamine (ManNAc), after which two pathways exist. One is the condensation of ManNAc and pyruvate into Neu5Ac through aldolase, and another is the condensation of ManNAc and phosphoenolpyruvate through synthetase.
Through homology search, we found several proteins that have identity with N-acetyl-D-neuraminic acid synthetase (neuB) from E. coli K1. These proteins were cloned, expressed and purified. The activities of aldolase, NeuB and AGE from different sources were determined. The results show that E. coli aldolase has the best activity:after 1 hour incubation at 37℃, the yield of Neu5Ac from ManNAc was 23.31% in whole-cell catalysis. NeuB from Neisseria meningitidis showed the best synthetase activity, with a 50.96% molar conversion after 30 minutes incubation at 37℃in whole-cell catalysis. BT0453 from Bacteroides thetaiotaomicron VPI-5482 showed the best AGE activity in that when coupled with E. coli aldolase in whole-cell catalysis,2.77% molar conversion from GlcNAc was observed after 4 hours incubation at 37℃in whole-cell catalysis.
Three E. coli chaperones were used as fusion tag to contruct chaperone-fusion protein expression vectors. Two proteins, A113695 and RB3348, were expressed in these vectors. Greatly improved solubility was observed for the expression of fusion proteins compared with the untagged protein expression. The trigger factor fused proteins showed the largest expression level among the chaperones. Bandscan analysis indicated that A113695 fused with trigger factor was a 95.5KD fusion protein, which was almost completely soluble, and the percentage of soluble fusion-protein in the total protein is 37.2%, higher than the percentage of untagged protein (7.1%). RB3348 fused with trigger factor was almost completely soluble too, and the percentage of soluble fusion-protein in the total protein is 53.0%, much higher than the percentage of untagged protein (4.4%).
The positive-selection vector is one kind of vectors that directly selecte recombinant clones through antibiotics or chemical compounds. The biggest advantage is that the cloning efficiency that can be up to 100%. It was reported that bla missed the C-terminal W290 codon lost its ampicillin-resistance activity, while cloning a target gene carrying the respective sequence to complement this amino acid residue at the C-terminus of Bla into the vector would result in restoring the protein's function. In this report, we construced improved positive-selection protein expression vectors pPAE and pPAE-MBP by cloning Bla that lacks of several C-terminus amino acids into an expression vector that harboring the restriction site of TEV protease. Cloning experiments with E. coli aldolase gene (nanA) and human renin binding protein (hPnBP) showed that both pPAE and pPAE-MBP have 100% cloning efficiency, and the genes successfully expressed in E. coli.
本研究通过同源搜索找到与已知神经氨酸合成酶具有较高同源性的蛋白,进而在大肠杆菌中进行克隆、表达和纯化。同时测定了不同来源的神经氨酸醛缩酶、神经氨酸合成酶以及N-酰基-D-葡萄糖胺2-异构酶的活性。结果表明来源于大肠杆菌的醛缩酶活性最高,以全细胞进行催化,37℃下反应1h后,神经氨酸的摩尔收率最高可以达到23.31%；来源于脑膜炎双球菌的神经氨酸合成酶在全细胞水平具有很高的合成酶活性,37℃下全细胞催化反应30分钟后Neu5Ac的摩尔收率最高可以达到50.96%；来源于多拟形杆菌的BT0453蛋白的异构酶活性较好,与大肠杆菌醛缩酶偶联反应,反应时间为4h后,神经氨酸的摩尔收率最高达到2.77%。
目前将来源自大肠杆菌的三种伴侣蛋白作为融合标签构建了伴侣蛋白融合表达载体。选取A113695和RB3348与伴侣蛋白基因融合表达。结果表明,目的基因与三种伴侣蛋白融合表达的重组蛋白可溶性明显增加,启动因子作为融合标签的融合蛋白表达量最高。BandScan软件分析表明,a113695基因与启动因子基因融合表达时,表达出95.5KD融合蛋白,所表达的融合蛋白几乎均为可溶性蛋白,其可溶性蛋白的表达量占总表达蛋白的比例为37.2%,高于a113695单独表达时的7.1%的比例；RB3348基因与启动因子基因融合表达时,可溶性蛋白的表达量占总表达蛋白的比例为53.0%,远高于单独表达时4.4%的比例。
正选择克隆载体是一类通过抗生素或化合物对重组克隆进行直接筛选的载体,最大优点是其可保证很高的克隆效率,有时可达100%。有报道显示β-内酰胺酶基因(bla)在缺失C端密码子(290位色氨酸密码子)时,将不能很好的表达氨苄青霉素抗性功能；而定向插入外源基因后将重建bla的C端密码子,进而使β-内酰胺酶重获氨苄青霉素抗性功能。本研究将色氨酸缺失的β-内酰胺酶基因克隆至带有TEV蛋白酶酶切位点的表达载体上,构建了正选择表达载体pPAE以及pPAE-MBP。选取大肠杆菌醛缩酶nanA和人源肾素结合蛋白hRnBP做克隆范例。实验结果证明pPAE和pPAE-MBP的克隆效率均达100%,同时所克隆的基因成功在大肠杆菌中表达。
N-Acetyl-D-neuraminic acid (Neu5Ac) is the key intermediate to chemical synthesis of Zanamivir, one of the two medicines used in the treatment of influenza. N-acetyl-D-neuraminic acid is currently made through enzymatic synthesis; the procedure consists of two steps:first, N-acetyl-D-glucosamine 2-epimerase (AGE) catalyzes the transformation of N-acetyl-D-glucosamine (GlcNAc) into N-acetyl-D-mannosamine (ManNAc), after which two pathways exist. One is the condensation of ManNAc and pyruvate into Neu5Ac through aldolase, and another is the condensation of ManNAc and phosphoenolpyruvate through synthetase.
Through homology search, we found several proteins that have identity with N-acetyl-D-neuraminic acid synthetase (neuB) from E. coli K1. These proteins were cloned, expressed and purified. The activities of aldolase, NeuB and AGE from different sources were determined. The results show that E. coli aldolase has the best activity:after 1 hour incubation at 37℃, the yield of Neu5Ac from ManNAc was 23.31% in whole-cell catalysis. NeuB from Neisseria meningitidis showed the best synthetase activity, with a 50.96% molar conversion after 30 minutes incubation at 37℃in whole-cell catalysis. BT0453 from Bacteroides thetaiotaomicron VPI-5482 showed the best AGE activity in that when coupled with E. coli aldolase in whole-cell catalysis,2.77% molar conversion from GlcNAc was observed after 4 hours incubation at 37℃in whole-cell catalysis.
Three E. coli chaperones were used as fusion tag to contruct chaperone-fusion protein expression vectors. Two proteins, A113695 and RB3348, were expressed in these vectors. Greatly improved solubility was observed for the expression of fusion proteins compared with the untagged protein expression. The trigger factor fused proteins showed the largest expression level among the chaperones. Bandscan analysis indicated that A113695 fused with trigger factor was a 95.5KD fusion protein, which was almost completely soluble, and the percentage of soluble fusion-protein in the total protein is 37.2%, higher than the percentage of untagged protein (7.1%). RB3348 fused with trigger factor was almost completely soluble too, and the percentage of soluble fusion-protein in the total protein is 53.0%, much higher than the percentage of untagged protein (4.4%).
The positive-selection vector is one kind of vectors that directly selecte recombinant clones through antibiotics or chemical compounds. The biggest advantage is that the cloning efficiency that can be up to 100%. It was reported that bla missed the C-terminal W290 codon lost its ampicillin-resistance activity, while cloning a target gene carrying the respective sequence to complement this amino acid residue at the C-terminus of Bla into the vector would result in restoring the protein's function. In this report, we construced improved positive-selection protein expression vectors pPAE and pPAE-MBP by cloning Bla that lacks of several C-terminus amino acids into an expression vector that harboring the restriction site of TEV protease. Cloning experiments with E. coli aldolase gene (nanA) and human renin binding protein (hPnBP) showed that both pPAE and pPAE-MBP have 100% cloning efficiency, and the genes successfully expressed in E. coli.
引文
1. Vann WF, Tavarez JJ, Crowley J et al. Purification and characterization of the Escherichia coli Kl neuB gene product N-acetylneuraminic acid synthetase. Glycobiology 1997; 7 (5):697-701.
2. Tabata K, Koizumi S, Endo T et al. Production of N-acetyl-D-neuraminic acid by coupling bacteria expressing N-acetyl-D-glucosamine 2-epimerase and N-acetyl-D-neuraminic acid synthetase. Enzyme and Microbial Technology 2002; 30 (3):327-33.
3. Suryanti V, Nelson A, Berry A. Cloning, over-expression, purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae. Protein Expr Purif 2003; 27 (2):346-56.
4. Sundaram AK, Pitts L, Muhammad K et al. Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni. Biochem J 2004; 383 (Pt 1):83-9.
5. Gunawan J, Simard D, Gilbert M et al. Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol. J Biol Chem 2005; 280 (5):3555-63.
6. Maru I, Ohnishi J, Ohta Y et al. Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine 2-epimerase and N-acetylneuraminate lyase. Carbohydr Res 1998; 306 (4):575-8.
7. Lee YC, Chien HC, Hsu WH. Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp. CHI N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase. J Biotechnol 2007; 129 (3):453-60.
8. Hu S, Chen J, Yang Z et al. Coupled bioconversion for preparation of N-acetyl-D-neuraminic acid using immobilized N-acetyl-D-glucosamine 2-epimerase and N-acetyl-D-neuraminic acid lyase. Appl Microbiol Biotechnol 2010; 85 (5):1383-91.
9. Hwang TS, Hung CH, Teo CF et al. Structural characterization of Escherichia coli sialic acid synthase. Biochem Biophys Res Commun 2002; 295 (1):167-73.
10. Hao J, Balagurumoorthy P, Sarilla S et al. Cloning, expression, and characterization of sialic acid synthases. Biochem Biophys Res Commun 2005; 338(3):1507-14.
11. Zimmermann V, Hennemann HG, Daussmann T et al. Modelling the reaction course of N-acetylneuraminic acid synthesis from N-acetyl-D-glucosamine-new strategies for the optimisation of neuraminic acid synthesis. Appl Microbiol Biotechnol 2007; 76 (3):597-605.
12. Mahmoudian M, Noble D, Drake CS et al. An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase. Enzyme Microb Technol 1997; 20 (5):393-400.
13. Wang TH, Chen YY, Pan HH et al. Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins. BMC Biotechnol 2009; 9:63.
14. Tao F, Zhang Y, Ma C et al. Biotechnological production and applications of N-acetyl-D-neuraminic acid:current state and perspectives. Appl Microbiol Biotechnol 2010; 87 (4):1281-9.
15. Zhang Y, Tao F, Du M et al. An efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system. Appl Microbiol Biotechnol 2010; 86 (2):481-9.
16. Takahashi S, Kumagai M, Shindo S et al. Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein). J Biochem 2000; 128 (6):951-6.
17. Denny PC, Denny PA, Allerton SE. Determination of sialic acid using 2-thiobarbituric acid in the absence of hazardous sodium arsenite. Clin Chim Acta 1983; 131 (3):333-6.
18. Ikura K, Kokubu T, Natsuka S et al. Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli. Prep Biochem Biotechnol 2002; 32 (2):189-205.
19. Nallamsetty S, Waugh DS. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat Protoc 2007; 2 (2):383-91.
20. Shen A, Lupardus PJ, Morell M et al. Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag. PLoS One 2009; 4 (12):e8119.
21. Tropea JE, Cherry S, Nallamsetty S et al. A generic method for the production of recombinant proteins in Escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag. Methods Mol Biol 2007; 363:1-19.
22. Waugh DS. Making the most of affinity tags. Trends Biotechnol 2005; 23 (6):316-20.
23. Kyratsous CA, Silverstein SJ, DeLong CR et al. Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli. Gene 2009; 440 (1-2):9-15.
24. Nishihara K, Kanemori M, Yanagi H et al. Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 2000; 66 (3):884-9.
25. Ho SN, Hunt HD, Horton RM et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77 (1):51-9.
26. Horton RM, Hunt HD, Ho SN et al. Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension. Gene 1989; 77 (1):61-8.
27. Heckman KL, Pease LR. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2007; 2 (4):924-32.
28. Dean D. A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene 1981; 15 (1):99-102.
29. Altenbuchner J, Viell P, Pelletier I. Positive selection vectors based on palindromic DNA sequences. Methods Enzymol 1992; 216:457-66.
30. Kast P. pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene 1994; 138 (1-2):109-14.
31. Choi YJ, Wang TT, Lee BH. Positive selection vectors. Crit Rev Biotechnol 2002; 22 (3):225-44.
32. Reddy M. Positive selection system for identification of recombinants using alpha-complementation plasmids. Biotechniques 2004; 37 (6):948-52.
33. Huang W, Petrosino J, Hirsch M et al. Amino acid sequence determinants of beta-lactamase structure and activity. J Mol Biol 1996; 258 (4):688-703.
34. Haag AF, Ostermeier C. Positive-selection vector for direct protein expression. Biotechniques 2009; 46 (6):453-7.
35. Blommel P G, Fox, B G. A combined approach to improving large-scale production of tobacco etch virus protease. Protein Expr Purif,2007,55 (1):53-68.
36. Rachel B, Kapust R B, Tozserl J, Fox J D, et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 2001,14 (12):993-1000.
37. Nallamsetty S, Waugh D S. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat Protoc 2007,2 (2):383-391.
38. Waugh D S. Making the most of affinity tags. Trends Biotechnol 2005,23 (6): 316-320.
39. Mahmoudian M, Noble D, Drake CS et al. An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase. Enzyme Microb Technol 1997; 20 (5):393-400.
40. Takahashi S, Kumagai M, Shindo S et al. Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein). J Biochem 2000; 128 (6):951-6.
41. Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press,1989, pp.16-34.
42.王立军,朱立,张恰轩,戈梅.鱼腥藻N-乙酰由-葡萄糖胺2-差向异构酶基因的克隆及其在大肠杆菌中的高效表达.沈阳药科大学学报,2008,25(12)：1007-1010
2. Tabata K, Koizumi S, Endo T et al. Production of N-acetyl-D-neuraminic acid by coupling bacteria expressing N-acetyl-D-glucosamine 2-epimerase and N-acetyl-D-neuraminic acid synthetase. Enzyme and Microbial Technology 2002; 30 (3):327-33.
3. Suryanti V, Nelson A, Berry A. Cloning, over-expression, purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae. Protein Expr Purif 2003; 27 (2):346-56.
4. Sundaram AK, Pitts L, Muhammad K et al. Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni. Biochem J 2004; 383 (Pt 1):83-9.
5. Gunawan J, Simard D, Gilbert M et al. Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol. J Biol Chem 2005; 280 (5):3555-63.
6. Maru I, Ohnishi J, Ohta Y et al. Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine 2-epimerase and N-acetylneuraminate lyase. Carbohydr Res 1998; 306 (4):575-8.
7. Lee YC, Chien HC, Hsu WH. Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp. CHI N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase. J Biotechnol 2007; 129 (3):453-60.
8. Hu S, Chen J, Yang Z et al. Coupled bioconversion for preparation of N-acetyl-D-neuraminic acid using immobilized N-acetyl-D-glucosamine 2-epimerase and N-acetyl-D-neuraminic acid lyase. Appl Microbiol Biotechnol 2010; 85 (5):1383-91.
9. Hwang TS, Hung CH, Teo CF et al. Structural characterization of Escherichia coli sialic acid synthase. Biochem Biophys Res Commun 2002; 295 (1):167-73.
10. Hao J, Balagurumoorthy P, Sarilla S et al. Cloning, expression, and characterization of sialic acid synthases. Biochem Biophys Res Commun 2005; 338(3):1507-14.
11. Zimmermann V, Hennemann HG, Daussmann T et al. Modelling the reaction course of N-acetylneuraminic acid synthesis from N-acetyl-D-glucosamine-new strategies for the optimisation of neuraminic acid synthesis. Appl Microbiol Biotechnol 2007; 76 (3):597-605.
12. Mahmoudian M, Noble D, Drake CS et al. An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase. Enzyme Microb Technol 1997; 20 (5):393-400.
13. Wang TH, Chen YY, Pan HH et al. Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins. BMC Biotechnol 2009; 9:63.
14. Tao F, Zhang Y, Ma C et al. Biotechnological production and applications of N-acetyl-D-neuraminic acid:current state and perspectives. Appl Microbiol Biotechnol 2010; 87 (4):1281-9.
15. Zhang Y, Tao F, Du M et al. An efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system. Appl Microbiol Biotechnol 2010; 86 (2):481-9.
16. Takahashi S, Kumagai M, Shindo S et al. Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein). J Biochem 2000; 128 (6):951-6.
17. Denny PC, Denny PA, Allerton SE. Determination of sialic acid using 2-thiobarbituric acid in the absence of hazardous sodium arsenite. Clin Chim Acta 1983; 131 (3):333-6.
18. Ikura K, Kokubu T, Natsuka S et al. Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli. Prep Biochem Biotechnol 2002; 32 (2):189-205.
19. Nallamsetty S, Waugh DS. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat Protoc 2007; 2 (2):383-91.
20. Shen A, Lupardus PJ, Morell M et al. Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag. PLoS One 2009; 4 (12):e8119.
21. Tropea JE, Cherry S, Nallamsetty S et al. A generic method for the production of recombinant proteins in Escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag. Methods Mol Biol 2007; 363:1-19.
22. Waugh DS. Making the most of affinity tags. Trends Biotechnol 2005; 23 (6):316-20.
23. Kyratsous CA, Silverstein SJ, DeLong CR et al. Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli. Gene 2009; 440 (1-2):9-15.
24. Nishihara K, Kanemori M, Yanagi H et al. Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 2000; 66 (3):884-9.
25. Ho SN, Hunt HD, Horton RM et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77 (1):51-9.
26. Horton RM, Hunt HD, Ho SN et al. Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension. Gene 1989; 77 (1):61-8.
27. Heckman KL, Pease LR. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2007; 2 (4):924-32.
28. Dean D. A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene 1981; 15 (1):99-102.
29. Altenbuchner J, Viell P, Pelletier I. Positive selection vectors based on palindromic DNA sequences. Methods Enzymol 1992; 216:457-66.
30. Kast P. pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene 1994; 138 (1-2):109-14.
31. Choi YJ, Wang TT, Lee BH. Positive selection vectors. Crit Rev Biotechnol 2002; 22 (3):225-44.
32. Reddy M. Positive selection system for identification of recombinants using alpha-complementation plasmids. Biotechniques 2004; 37 (6):948-52.
33. Huang W, Petrosino J, Hirsch M et al. Amino acid sequence determinants of beta-lactamase structure and activity. J Mol Biol 1996; 258 (4):688-703.
34. Haag AF, Ostermeier C. Positive-selection vector for direct protein expression. Biotechniques 2009; 46 (6):453-7.
35. Blommel P G, Fox, B G. A combined approach to improving large-scale production of tobacco etch virus protease. Protein Expr Purif,2007,55 (1):53-68.
36. Rachel B, Kapust R B, Tozserl J, Fox J D, et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 2001,14 (12):993-1000.
37. Nallamsetty S, Waugh D S. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat Protoc 2007,2 (2):383-391.
38. Waugh D S. Making the most of affinity tags. Trends Biotechnol 2005,23 (6): 316-320.
39. Mahmoudian M, Noble D, Drake CS et al. An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase. Enzyme Microb Technol 1997; 20 (5):393-400.
40. Takahashi S, Kumagai M, Shindo S et al. Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein). J Biochem 2000; 128 (6):951-6.
41. Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press,1989, pp.16-34.
42.王立军,朱立,张恰轩,戈梅.鱼腥藻N-乙酰由-葡萄糖胺2-差向异构酶基因的克隆及其在大肠杆菌中的高效表达.沈阳药科大学学报,2008,25(12)：1007-1010