人溶菌酶N端与Exendin-4嵌合蛋白的原核表达、纯化及鉴定
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摘要
目的：将目前公认对Ⅱ型糖尿病有良好治疗效果、已获得临床应用的多肽药物exendin-4,与另一个可用于防治糖尿病慢性并发症的人溶菌酶N端多肽串联并进行原核表达、纯化及鉴定。该设计的嵌合多肽可被人体内自然存在的凝血酶和二肽基肽酶水解,释放出发挥不同药理作用的人溶菌酶N端多肽和exendin-4,从不同环节发挥治疗糖尿病的作用,为探索一种新型双靶向治疗糖尿病的多肽药物奠定实验基础。
方法：通过RT-PCR从人外周血获得人溶菌酶N端74个氨基酸的基因序列,然后利用重组PCR技术将其与人工合成的exendin-4基因序列相连接,连接序列为一段可被凝血酶和二肽基肽酶分别切割的短肽基因序列。以嵌合基因hLYZ(N74)-exendin-4与质粒pET-32a(+)构建原核表达载体,转化大肠杆菌BL21(DE3)并诱导表达,SDS-PAGE和Western blotting鉴定表达的蛋白。经表达条件优化,大量制备重组蛋白,变性条件下进行镍离子亲和层析纯化。对纯化后的重组蛋白进行复性,用肠激酶切割复性蛋白以获得目的多肽。重组蛋白经凝血酶切割和HPLC分离后,以质谱鉴定所获得的多肽水解片段。
结果：重组质粒pET-32a／hLYZ(N74)-Ex4构建正确,序列分析结果与预期完全相符；重组蛋白获得高效表达,37℃诱导4h、IPTG浓度为0.6mmol／L时表达量最高,约占菌体蛋白总量的30%；SDS-PAGE结果显示重组蛋白分子量约为30kD,Western blotting检测表明重组蛋白为单一清晰条带,与抗His-tag单克隆抗体有较强的特异性反应；重组蛋白表达主要以包涵体形式存在,经亲和层析纯化后获得了高纯度的重组蛋白；对重组蛋白的透析复性研究表明,4℃条件下、蛋白浓度为50μg／mL、添加L-精氨酸和GSH/GSSG时,蛋白复性率较高,达23.8%；复性蛋白经肠激酶切割可获得较好纯度的目的嵌合多肽；重组蛋白经凝血酶切割,检测到相对分子质量为4341.0的exendin-4片段。
结论：hLYZ(N74)-exendin-4嵌合蛋白的原核表达质粒构建成功,并实现了重组蛋白的高效表达。重组蛋白能够被肠激酶切割产生目的多肽,设计引入的凝血酶切割位点有效。
Objectives:Exendin-4 is a polypeptide of 39 amino acids which has recently been clinically used and demonstrated to have good therapeutic efficacy for type 2 diabetes. Human lysozyme N-terminal polypeptide has been shown to be able to bind AGEs and to increase the clearance of AGEs from body. Thus, this peptide may delay or prevent the development of diabetic complications. In this study, our aim was to link these two polypeptide genes by using a linker sequence which encodes for an amino acid sequence recognized by thrombin and dipeptidyl peptidase IV, so that the chimeric peptide can be cleaved into lysozyme N-terminal fragment and exendin-4 by enzymes in the body, and both of these products may play different roles in the treatment of diabetes. The expression, purification and identification of this chimeric peptide in E. coli will provide an experimental foundation for development of a new type of dual-targeting protein drugs.
Methods:The gene sequence for human lysozyme N-terminal fragment(1-74aa) was prepared by RT-PCR from human leucocytes. This sequence was then linked to exendin-4 gene sequence by using recombinant PCR. The linker sequence between lysozyme fragment and exendin-4 contained a site recognized by thrombin and another site by dipeptidyl peptidase IV. HLYZ(N74)-exendin-4 gene was cloned into a prokaryotic expression vector, pET-32a(+), and then transformed into E. coli BL21(DE3). After induced with IPTG, the recombinant protein was identified by SDS-PAGE and Western blotting. After optimization of the expression condition, a high expression level of the recombinant protein were achieved. The polypeptide of interest was purified by Ni2+ affinity chromatography under denaturing conditions, followed by renaturation of the recombinant protein by dialysis, and digestion with enterokinase to release the chimeric peptide. After cleavage of the recombinant protein by thrombin, the hydrolisis fragments were finally separated by HPLC and identified by mass spectrometry.
Results:DNA sequence analysis showed that the recombinant plasmid pET-32a/hLYZ(N74)-Ex4 was constructed correctly. The recombinant protein was highly expressed, and reached a maximum level of about 30% of total somatic proteins after induction with 0.6mmol/L IPTG at 37℃for 4h. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was about 30kD. Western blotting analysis suggested that the recombinant protein had a strong reaction with anti-His-tag antibody, exhibiting as a single clear band. The recombinant protein was expressed mainly in the form of inclusion bodies and could be highly purified by Ni2+ affinity chromatography. The recombinant protein was renatured by dialysis, and the refolding conditions was found to be 50μg/mL of recombinant proteins,4℃, and with addition of L-Arg and GSH/GSSG in the dialysate, which contributed to a higher level of yield, at about 23.8%. Our results also showed that the recombinant protein was cleaved into two fragments by enterokinase, the Trx-His tag and the chimeric peptide hLYZ(N74)-exendin-4. After cleavage of the recombinant protein by thrombin, the exendin-4 fragment was detected at the molecular weight of 4341.0 by mass spectrometry.
Conclusion:The prokaryotic expression vector for hLYZ(N74)-exendin-4 fusion gene was successfully constructed, and the recombinant protein was highly expressed in E. coli. This recombinant protein can produce the chimeric peptide, hLYZ(N74)-exendin-4, after digestion with enterokinase, which contains a thrombin hydrolytic site between the human lysozyme N-terminal fragment and exendin-4.
方法：通过RT-PCR从人外周血获得人溶菌酶N端74个氨基酸的基因序列,然后利用重组PCR技术将其与人工合成的exendin-4基因序列相连接,连接序列为一段可被凝血酶和二肽基肽酶分别切割的短肽基因序列。以嵌合基因hLYZ(N74)-exendin-4与质粒pET-32a(+)构建原核表达载体,转化大肠杆菌BL21(DE3)并诱导表达,SDS-PAGE和Western blotting鉴定表达的蛋白。经表达条件优化,大量制备重组蛋白,变性条件下进行镍离子亲和层析纯化。对纯化后的重组蛋白进行复性,用肠激酶切割复性蛋白以获得目的多肽。重组蛋白经凝血酶切割和HPLC分离后,以质谱鉴定所获得的多肽水解片段。
结果：重组质粒pET-32a／hLYZ(N74)-Ex4构建正确,序列分析结果与预期完全相符；重组蛋白获得高效表达,37℃诱导4h、IPTG浓度为0.6mmol／L时表达量最高,约占菌体蛋白总量的30%；SDS-PAGE结果显示重组蛋白分子量约为30kD,Western blotting检测表明重组蛋白为单一清晰条带,与抗His-tag单克隆抗体有较强的特异性反应；重组蛋白表达主要以包涵体形式存在,经亲和层析纯化后获得了高纯度的重组蛋白；对重组蛋白的透析复性研究表明,4℃条件下、蛋白浓度为50μg／mL、添加L-精氨酸和GSH/GSSG时,蛋白复性率较高,达23.8%；复性蛋白经肠激酶切割可获得较好纯度的目的嵌合多肽；重组蛋白经凝血酶切割,检测到相对分子质量为4341.0的exendin-4片段。
结论：hLYZ(N74)-exendin-4嵌合蛋白的原核表达质粒构建成功,并实现了重组蛋白的高效表达。重组蛋白能够被肠激酶切割产生目的多肽,设计引入的凝血酶切割位点有效。
Objectives:Exendin-4 is a polypeptide of 39 amino acids which has recently been clinically used and demonstrated to have good therapeutic efficacy for type 2 diabetes. Human lysozyme N-terminal polypeptide has been shown to be able to bind AGEs and to increase the clearance of AGEs from body. Thus, this peptide may delay or prevent the development of diabetic complications. In this study, our aim was to link these two polypeptide genes by using a linker sequence which encodes for an amino acid sequence recognized by thrombin and dipeptidyl peptidase IV, so that the chimeric peptide can be cleaved into lysozyme N-terminal fragment and exendin-4 by enzymes in the body, and both of these products may play different roles in the treatment of diabetes. The expression, purification and identification of this chimeric peptide in E. coli will provide an experimental foundation for development of a new type of dual-targeting protein drugs.
Methods:The gene sequence for human lysozyme N-terminal fragment(1-74aa) was prepared by RT-PCR from human leucocytes. This sequence was then linked to exendin-4 gene sequence by using recombinant PCR. The linker sequence between lysozyme fragment and exendin-4 contained a site recognized by thrombin and another site by dipeptidyl peptidase IV. HLYZ(N74)-exendin-4 gene was cloned into a prokaryotic expression vector, pET-32a(+), and then transformed into E. coli BL21(DE3). After induced with IPTG, the recombinant protein was identified by SDS-PAGE and Western blotting. After optimization of the expression condition, a high expression level of the recombinant protein were achieved. The polypeptide of interest was purified by Ni2+ affinity chromatography under denaturing conditions, followed by renaturation of the recombinant protein by dialysis, and digestion with enterokinase to release the chimeric peptide. After cleavage of the recombinant protein by thrombin, the hydrolisis fragments were finally separated by HPLC and identified by mass spectrometry.
Results:DNA sequence analysis showed that the recombinant plasmid pET-32a/hLYZ(N74)-Ex4 was constructed correctly. The recombinant protein was highly expressed, and reached a maximum level of about 30% of total somatic proteins after induction with 0.6mmol/L IPTG at 37℃for 4h. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was about 30kD. Western blotting analysis suggested that the recombinant protein had a strong reaction with anti-His-tag antibody, exhibiting as a single clear band. The recombinant protein was expressed mainly in the form of inclusion bodies and could be highly purified by Ni2+ affinity chromatography. The recombinant protein was renatured by dialysis, and the refolding conditions was found to be 50μg/mL of recombinant proteins,4℃, and with addition of L-Arg and GSH/GSSG in the dialysate, which contributed to a higher level of yield, at about 23.8%. Our results also showed that the recombinant protein was cleaved into two fragments by enterokinase, the Trx-His tag and the chimeric peptide hLYZ(N74)-exendin-4. After cleavage of the recombinant protein by thrombin, the exendin-4 fragment was detected at the molecular weight of 4341.0 by mass spectrometry.
Conclusion:The prokaryotic expression vector for hLYZ(N74)-exendin-4 fusion gene was successfully constructed, and the recombinant protein was highly expressed in E. coli. This recombinant protein can produce the chimeric peptide, hLYZ(N74)-exendin-4, after digestion with enterokinase, which contains a thrombin hydrolytic site between the human lysozyme N-terminal fragment and exendin-4.
引文
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