重组大肠杆菌高效生产具生物活性的人β-防御素和牛肠激酶的研究
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摘要
人β-防御素是近来年发现的具有广谱抗菌活性并在机体抵御外来微生物入侵中起防御作用的一类阳离子抗菌肽。肠激酶是哺乳动物肠道内的一种丝氨酸蛋白水解酶，能够特异性的识别(Asp)_4-Lys序列，起到把胰蛋白酶原转变成胰蛋白酶的作用。
本论文研究了使用基因工程技术生产人β-防御素3及4(HBD3、HBD4)和牛肠激酶轻链(BEKLC)的方法。建立了从获得HBD3、HBD4和牛肠激酶轻链基因、载体构建、表达优化、产物分离纯化、BEKLC包涵体复性，直到生物活性测定等的完整工艺。
经过密码子优化全基因合成了HBD3及4的基因，构建了大肠杆菌融合表达HBD3及4的基因工程菌，通过对摇瓶表达条件的研究，获得了如下的优化培养条件：在MBL培养基，培养温度为34℃，在对数生长中期添加终浓度为0.4 mM的IPTG进行诱导的情况下，HBD3和HBD4重组菌分别诱导8 h和6h终止发酵，首次实现了融合蛋白的高水平表达。HBD3融合蛋白的产量为2.55g／L，HBD4融合蛋白的产量达到2.68 g／L。
经过密码子优化全基因合成了牛肠激酶轻链基因(sBEKLC)，采用质粒pET-39b(+)构建了融合表达载体，成功地在大肠杆菌中进行了表达，融合蛋白产量达到151.2 mg／L，i.e.80.6 mg／L sBEKLC。使用渗透冲击技术从细胞周质中提取sBEKLC，经过亲和层析可从每升发酵液中得到6.8mg sBEKLC，在类似的研究工作中处于较高的水平。经检测，sBEKLC具有生物活性。
部分表达的sBEKLC融合蛋白为不可溶，应用镍离子金属螯地合层析对包涵体进行了纯化和复性研究，优化的复性条件为：50 mM Tris-HCl(pH 8.0)，0.3 MNaCl，2 Murea，10 mM Imidazole，3 mM GSH，0.6 mM GSSG条件下，复性6 h结束，复性产物具有生物活性。
将自制的肠激酶轻链成功地用于人防御素融合蛋白的切割以释放出有生物活性的成熟的产物。对重组菌生产的人β-防御素-3及4的分离纯化工艺进行了研究，分别建立了成熟HBD3总收率为41％、纯度为92％和成熟HBD4总收率为44％、纯度为95％的分离提纯工艺；测定了成熟产物的生物活性，所得重组蛋白经测定具有抑菌活性。
Human β-defensins are a group of cationic antibiotic peptides with broad antibacterial spectrum discovered in recent years, which play important roles in the self-defense against microbial invasion. Enterokinase is a serine Proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)4-Lys and converts trypsinogen into its active form trypsin.
In this dissertation, a process of recombinant expression of human β-defensin-3 and -4(HBD3, HBD4) as well as bovine enterokinase light chain (BEKLC) was proposed, which includes the codon optimization of the genes, the construction of expression vectors, the optimization of the expression conditions, the purification of the target protein, refolding the inclusion bodies of BEKLC and bioactivity determination of the product.
The codon optimized sequences coding HBD3 and HBD4 gene were synthesized, which were fused with TrxA to construct the expression vector. The resulting vector was finally transformed into E. coli BL21 (DE3) for expression. The optimal culture conditions of HBD3 and HBD4 production strain were determined as following: cultivation at 28℃ in MBL medium, induction at middle stage of exponential growth with 0.4 mM IPTG, and post-induction expression for 8 h of HBD3 and 6 h of HBD4. The high level expressions of HBD3 and HBD4 fusion protein were realized and never reported before. The volumetric productivity of fusion proteins were 2.55 g/l and 2.68 g/l for HBD3 and HBD4, respectively.
A codon optimized sequence coding light chain of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector. Then the plasmid was transformed into E. coli BL21 (DE3) for expression, the volumetric productivity of fusion protein reached 151.2 mg/L, i.e. 80.6mg/L sBEKLC. The cold osmotic shock technique was successfully used to extract sulale sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8mg of sBEKLC was purified from 1 liter fermentation broth and the enzyme activity of sBEKLC was well demonstrated.
The inclusion bodies of sBEKLC fusion protein were purified under denaturing
conditions and refolded by using refolding buffer in Ni-NTA column. The optimal refolding conditions were determined as following: 50 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 2 M urea, 10 mM Imidazole, 3 mM GSH, 0.6 mM GSSG and refolding for 6 h. The enzyme activity of the product was detected.
The self-made sBEKLC was successfully applied to split fusion protein and to release the mature bioactive HBD3 or HBD4. The purification procedure was established to obtain the recombinant HBD3 and HBD4. The overall recovery ratio of HBD3 was 41% with a purity of 92%, and which of HBD4 reached 44% with a purity of 95%. The antimicrobial activitis of the products were well determined.
本论文研究了使用基因工程技术生产人β-防御素3及4(HBD3、HBD4)和牛肠激酶轻链(BEKLC)的方法。建立了从获得HBD3、HBD4和牛肠激酶轻链基因、载体构建、表达优化、产物分离纯化、BEKLC包涵体复性，直到生物活性测定等的完整工艺。
经过密码子优化全基因合成了HBD3及4的基因，构建了大肠杆菌融合表达HBD3及4的基因工程菌，通过对摇瓶表达条件的研究，获得了如下的优化培养条件：在MBL培养基，培养温度为34℃，在对数生长中期添加终浓度为0.4 mM的IPTG进行诱导的情况下，HBD3和HBD4重组菌分别诱导8 h和6h终止发酵，首次实现了融合蛋白的高水平表达。HBD3融合蛋白的产量为2.55g／L，HBD4融合蛋白的产量达到2.68 g／L。
经过密码子优化全基因合成了牛肠激酶轻链基因(sBEKLC)，采用质粒pET-39b(+)构建了融合表达载体，成功地在大肠杆菌中进行了表达，融合蛋白产量达到151.2 mg／L，i.e.80.6 mg／L sBEKLC。使用渗透冲击技术从细胞周质中提取sBEKLC，经过亲和层析可从每升发酵液中得到6.8mg sBEKLC，在类似的研究工作中处于较高的水平。经检测，sBEKLC具有生物活性。
部分表达的sBEKLC融合蛋白为不可溶，应用镍离子金属螯地合层析对包涵体进行了纯化和复性研究，优化的复性条件为：50 mM Tris-HCl(pH 8.0)，0.3 MNaCl，2 Murea，10 mM Imidazole，3 mM GSH，0.6 mM GSSG条件下，复性6 h结束，复性产物具有生物活性。
将自制的肠激酶轻链成功地用于人防御素融合蛋白的切割以释放出有生物活性的成熟的产物。对重组菌生产的人β-防御素-3及4的分离纯化工艺进行了研究，分别建立了成熟HBD3总收率为41％、纯度为92％和成熟HBD4总收率为44％、纯度为95％的分离提纯工艺；测定了成熟产物的生物活性，所得重组蛋白经测定具有抑菌活性。
Human β-defensins are a group of cationic antibiotic peptides with broad antibacterial spectrum discovered in recent years, which play important roles in the self-defense against microbial invasion. Enterokinase is a serine Proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)4-Lys and converts trypsinogen into its active form trypsin.
In this dissertation, a process of recombinant expression of human β-defensin-3 and -4(HBD3, HBD4) as well as bovine enterokinase light chain (BEKLC) was proposed, which includes the codon optimization of the genes, the construction of expression vectors, the optimization of the expression conditions, the purification of the target protein, refolding the inclusion bodies of BEKLC and bioactivity determination of the product.
The codon optimized sequences coding HBD3 and HBD4 gene were synthesized, which were fused with TrxA to construct the expression vector. The resulting vector was finally transformed into E. coli BL21 (DE3) for expression. The optimal culture conditions of HBD3 and HBD4 production strain were determined as following: cultivation at 28℃ in MBL medium, induction at middle stage of exponential growth with 0.4 mM IPTG, and post-induction expression for 8 h of HBD3 and 6 h of HBD4. The high level expressions of HBD3 and HBD4 fusion protein were realized and never reported before. The volumetric productivity of fusion proteins were 2.55 g/l and 2.68 g/l for HBD3 and HBD4, respectively.
A codon optimized sequence coding light chain of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector. Then the plasmid was transformed into E. coli BL21 (DE3) for expression, the volumetric productivity of fusion protein reached 151.2 mg/L, i.e. 80.6mg/L sBEKLC. The cold osmotic shock technique was successfully used to extract sulale sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8mg of sBEKLC was purified from 1 liter fermentation broth and the enzyme activity of sBEKLC was well demonstrated.
The inclusion bodies of sBEKLC fusion protein were purified under denaturing
conditions and refolded by using refolding buffer in Ni-NTA column. The optimal refolding conditions were determined as following: 50 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 2 M urea, 10 mM Imidazole, 3 mM GSH, 0.6 mM GSSG and refolding for 6 h. The enzyme activity of the product was detected.
The self-made sBEKLC was successfully applied to split fusion protein and to release the mature bioactive HBD3 or HBD4. The purification procedure was established to obtain the recombinant HBD3 and HBD4. The overall recovery ratio of HBD3 was 41% with a purity of 92%, and which of HBD4 reached 44% with a purity of 95%. The antimicrobial activitis of the products were well determined.
引文
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1 Fang X, Peng L, Xu ZN, Wu JM, and Cen PL. Cloning and expression of human beta-defensin-2 gene in Escherichia coli. Protein Pept. Lett. 2002, 9: 31-37.
2 Peng L, Xu ZN, Fang XM, Wang F, Yang S, and Cen PL. Preferential codons enhancing the expression level of human beta-defensin-2, Protein Pept. Lett. 2004, 11:339-344.
3 Peng L, Xu ZN, Fang XM, Wang F, and Cen PL. High-level expression of soluble human beta-defensin-2 in E. coli. Process Biochem. 2004, 39: 2199-2205.
4 Wang F, Fang XM, Xu ZN, Peng L, and Cen PL. Fusion expression of human beta-defensin-2 from multiple jointed genes in E. coli. Prep. Biochem. Biotech. 2004, 34, 215-225.
5 Xu ZN, Peng L, Zhong ZX, Fang XM, and Cen PL. High-level expression of a soluble functional antimicrobial peptide, human beta-defensin 2, in Escherichia coli. Biotechnol Prog. 2006, 22(2): 382-6.
6 Zhong ZX, Xu ZN, Peng L, Huang L, Fang XM, and Cen PL. Tandem repeat mHBD2 gene enhance the soluble fusion expression of HBD2 in Escherichia coli. Applied microbiology and biotechnology. 2006, in press.
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11 Hu XY, Shi QW, Yang T, and Jackowski G.. Specific replacement of consecutive AGG codons results in high-level expression of human cardiac troponin T in Escherichia coli. Protein Expres Purif. 1996, 7: 289-293.
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2 Liepnieks JJ and Light A. The preparation and properties of bovine enterokinase. The Journal of biological chemistry. 1979, 254(5): 1677-1683.
3 Light A and Fonseea P. The preparation and properties of the catalytic subunit of bovine enterokinase. The Journal of biological chemistry. 1984, 259(21): 13195-13198.
4 Lu D, Yuan X, Zheng XL, and Sadler JE. Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain. Journal of biological Chemistry, 1997, 272: 31293-31300.
5 Kane JF. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 1995, 6: 494-500.
6 Spanjaard RA, Chen K, Walker JR, and van Duin J. Frameshift suppression at tandem AGA and AGG. codons by cloned tRNA genes: Assigning a codon to argU tRNA and T4 tRNAarg. Nucleic Acids Res 1990; 18: 5031-5036.
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