重组脯氨酰内肽酶提取方法改进及活性研究
|
摘要
脯氨酰内肽酶具有水解多肽中脯氨酰残基的能力,具有治疗乳糜泻的潜力,构建高表达量的重组脯氨酰内肽酶、建立保持其高活性的提取方法对于脯氨酰内肽酶的药物开发至关重要。该研究经密码子优化、基因合成,构建了重组鞘氨醇单胞菌脯氨酰内肽酶(SCPEP)的大肠杆菌BL21(DE3)工程菌。探索建立改进型渗透休克法提取SCPEP,经Ni-NTA纯化、超滤浓缩得到产物。采用分光光度法基于底物Suc-Ala-Pro-pNA评价SCPEP的活性,同时与高压均质法提取的产物进行对比研究。结果显示,在pH 6. 0和pH 7. 0的反应体系中,采用改进型渗透休克法提取的SCPEP水解Suc-Ala-Pro-pNA的kcat/KM分别为(56. 58±12. 00)(mmol/L)~(-1)·s~(-1)和(55. 82±15. 33)(mmol/L)~(-1)·s~(-1),而高压均质法提取的SCPEP水解Suc-Ala-Pro-pNA的kcat/KM分别为(24. 78±10. 63)(mmol/L)~(-1)·s~(-1)和(48. 69±13. 79)(mmol/L)~(-1)·s~(-1),改进型渗透休克法提取的SCPEP在pH6. 0和pH 7. 0下的活性比高压均质法提取的SCPEP高2. 30和1. 15倍,故改进型渗透休克法有利于提取的SCPEP保持更高的活性。
Prolyl endopeptidase( PEP) has an ability to hydrolyze prolyl residues in polypeptides; therefore,it has the potential to treat celiac disease. Optimizing the recombinant expression and purification methods of PEP is very important to the drug development of prolyl endopeptidase. The purpose of this study is to construct a high-expression recombinant SCPEP by molecular biology techniques and to explore a new extraction method to maintain the high activity of SCPEP. And the effect of the new extraction method-modified osmotic shock method and traditional high-pressure homogenization extraction method on the activity of recombinant SCPEP is compared and analyzed. In this study,recombinant Escherichia coli BL21( DE3) engineered strain were constructed by codon optimization and gene synthesis. In the exploration of the establishment of a new extraction method,this study reduced the centrifugal speed of the traditional osmotic shock method and established a modified osmotic shock method. Then SCPEP was extracted by modified osmotic shock method and high pressure homogenization. At last SCPEP was purified by Ni-NTA and concentrated by ultrafiltration. The activity of SCPEP extracted by modified osmotic shock method was evaluated by spectrophotometry based on the substrate Suc-Ala-Pro-pNA,and compared with the products extracted by high pressure homogenization. The results showed that at pH 6. 0 and pH 7. 0,the kcat/KMof SCPEP extracted by modified osmotic shock method was( 56. 58 ± 12. 00)( mmol/L)~(-1)·s~(-1) and( 55. 82 ± 15. 33)( mmol/L)~(-1)·s~(-1),respectively; while the kcat/KMof SCPEP extracted by high pressure homogenization method was( 24. 78 ± 10. 63)( mmol/L)~(-1)·s~(-1) and( 48. 69 ± 13. 79)( mmol/L)~(-1)·s~(-1),respectively. SCPEP extracted by modified osmotic shock method has 2. 30 times and1. 15 times higher catalytic activity at pH 6. 0 and pH 7. 0 than SCPEP extracted by high pressure homogenization. In summary,this study successfully constructed a high expression recombinant wild-type SCPEP,and compared the two methods of extracting recombinant SCPEP,and optimized the centrifugal conditions of the osmotic shock method. From the perspective of production,this study optimized the osmotic shock extraction method,which made it easier to carry out large-scale industrial production. From the perspective of maintaining activity,the modified osmotic shock method is advantageous for the extracted SCPEP to maintain higher activity.
Prolyl endopeptidase( PEP) has an ability to hydrolyze prolyl residues in polypeptides; therefore,it has the potential to treat celiac disease. Optimizing the recombinant expression and purification methods of PEP is very important to the drug development of prolyl endopeptidase. The purpose of this study is to construct a high-expression recombinant SCPEP by molecular biology techniques and to explore a new extraction method to maintain the high activity of SCPEP. And the effect of the new extraction method-modified osmotic shock method and traditional high-pressure homogenization extraction method on the activity of recombinant SCPEP is compared and analyzed. In this study,recombinant Escherichia coli BL21( DE3) engineered strain were constructed by codon optimization and gene synthesis. In the exploration of the establishment of a new extraction method,this study reduced the centrifugal speed of the traditional osmotic shock method and established a modified osmotic shock method. Then SCPEP was extracted by modified osmotic shock method and high pressure homogenization. At last SCPEP was purified by Ni-NTA and concentrated by ultrafiltration. The activity of SCPEP extracted by modified osmotic shock method was evaluated by spectrophotometry based on the substrate Suc-Ala-Pro-pNA,and compared with the products extracted by high pressure homogenization. The results showed that at pH 6. 0 and pH 7. 0,the kcat/KMof SCPEP extracted by modified osmotic shock method was( 56. 58 ± 12. 00)( mmol/L)~(-1)·s~(-1) and( 55. 82 ± 15. 33)( mmol/L)~(-1)·s~(-1),respectively; while the kcat/KMof SCPEP extracted by high pressure homogenization method was( 24. 78 ± 10. 63)( mmol/L)~(-1)·s~(-1) and( 48. 69 ± 13. 79)( mmol/L)~(-1)·s~(-1),respectively. SCPEP extracted by modified osmotic shock method has 2. 30 times and1. 15 times higher catalytic activity at pH 6. 0 and pH 7. 0 than SCPEP extracted by high pressure homogenization. In summary,this study successfully constructed a high expression recombinant wild-type SCPEP,and compared the two methods of extracting recombinant SCPEP,and optimized the centrifugal conditions of the osmotic shock method. From the perspective of production,this study optimized the osmotic shock extraction method,which made it easier to carry out large-scale industrial production. From the perspective of maintaining activity,the modified osmotic shock method is advantageous for the extracted SCPEP to maintain higher activity.
引文
[1] Lebwohl B,Sanders DS,Green PHR. Coeliac disease[J]. Lancet,2018,391:70-81.
[2] Kamran R,Justine B,Alison P,et al. Gluten-free diet indications,safety,quality,labels,and challenges[J]. Nutrients,2017,9(8):846-.
[3] Bascunán KA,Vespa MC,Araya M. Celiac disease:understanding the gluten-free diet[J]. European J Nutrit,2017,56(2):449-459.
[4] Bethune MT,Khosla C. Oral enzyme therapy for celiac sprue[J].Methods Enzymol,2012,502:241-271.
[5] Shan L,Marti T,Sollid L,et al. Comparative biochemical analysis of three bacterial prolyl endopeptidases:implications for coeliac sprue[J]. Biochem J,2004,383(2):311-318.
[6] Tye-Din JA,Anderson RP,Ffrench RA,et al. The effects of ALV003 pre-digestion of gluten on immune response and symptoms in celiac disease in vivo[J]. Clin Immunol,2010,134(3):289-295.
[7] Syage JA,Murray JA,Green PHR,et al. Latiglutenase improves symptoms in seropositive celiac disease patients while on a glutenfree diet[J]. Digest Dis Sci,2017,62(9):1-5.
[8] Gass J,Khosla C. Prolyl endopeptidases[J]. Cellular Molecular Life Sci,2007,64(3):345-355.
[9] Kabashima T,Fujii M,Meng Y,et al. Prolyl endopeptidase from sphingomonas capsulata:isolation and characterization of the enzyme and nucleotide sequence of the gene[J]. Archives Biochem Biophys,1998,358(1):0-148.
[10] Kupfer SS,Jabri B. Pathophysiology of celiac disease[J]. Gastrointest Endoscopy Clinics North America,2012,22(4):639-660.
[11]李振国,徐明波,牛罡,在大肠杆菌周质表达重组蛋白的研究进展[J].药物生物技术,2011,18(1):73-76.
[12] Leite Júnior Bruno Ricardo de Castro,Lima TAA,Marcelo C,et al. High pressure homogenization of porcine pepsin protease:effects on enzyme activity,stability,milk coagulation profile and gel development[J]. PLOS ONE,2015,10(5):e0125061.
[13]李夏兰,蔡婀娜,曾明荣.温和化学渗透法破碎酵母细胞的研究[J].药物生物技术,2000,7(1):23-27.
[14] Dos Santos JG,Cristianini M,Sato HH. Modification of enzymes by use of high-pressure homogenization[J]. Food Res Internat,2018:S0963996918302825.
[2] Kamran R,Justine B,Alison P,et al. Gluten-free diet indications,safety,quality,labels,and challenges[J]. Nutrients,2017,9(8):846-.
[3] Bascunán KA,Vespa MC,Araya M. Celiac disease:understanding the gluten-free diet[J]. European J Nutrit,2017,56(2):449-459.
[4] Bethune MT,Khosla C. Oral enzyme therapy for celiac sprue[J].Methods Enzymol,2012,502:241-271.
[5] Shan L,Marti T,Sollid L,et al. Comparative biochemical analysis of three bacterial prolyl endopeptidases:implications for coeliac sprue[J]. Biochem J,2004,383(2):311-318.
[6] Tye-Din JA,Anderson RP,Ffrench RA,et al. The effects of ALV003 pre-digestion of gluten on immune response and symptoms in celiac disease in vivo[J]. Clin Immunol,2010,134(3):289-295.
[7] Syage JA,Murray JA,Green PHR,et al. Latiglutenase improves symptoms in seropositive celiac disease patients while on a glutenfree diet[J]. Digest Dis Sci,2017,62(9):1-5.
[8] Gass J,Khosla C. Prolyl endopeptidases[J]. Cellular Molecular Life Sci,2007,64(3):345-355.
[9] Kabashima T,Fujii M,Meng Y,et al. Prolyl endopeptidase from sphingomonas capsulata:isolation and characterization of the enzyme and nucleotide sequence of the gene[J]. Archives Biochem Biophys,1998,358(1):0-148.
[10] Kupfer SS,Jabri B. Pathophysiology of celiac disease[J]. Gastrointest Endoscopy Clinics North America,2012,22(4):639-660.
[11]李振国,徐明波,牛罡,在大肠杆菌周质表达重组蛋白的研究进展[J].药物生物技术,2011,18(1):73-76.
[12] Leite Júnior Bruno Ricardo de Castro,Lima TAA,Marcelo C,et al. High pressure homogenization of porcine pepsin protease:effects on enzyme activity,stability,milk coagulation profile and gel development[J]. PLOS ONE,2015,10(5):e0125061.
[13]李夏兰,蔡婀娜,曾明荣.温和化学渗透法破碎酵母细胞的研究[J].药物生物技术,2000,7(1):23-27.
[14] Dos Santos JG,Cristianini M,Sato HH. Modification of enzymes by use of high-pressure homogenization[J]. Food Res Internat,2018:S0963996918302825.