|Table of Contents|

Chromosome-based TEV Protease Overexpression(PDF)

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

Issue:
2014年03期
Page:
95-
Research Field:
生命科学
Publishing date:

Info

Title:
Chromosome-based TEV Protease Overexpression
Author(s):
Li Ling; Ling Wen; Shi Mudan; Shang Guangdong
School of Life Sciences,Nanjing Normal University,Jiangsu Engineering and Technology Research Center for Microbiology,Jiangsu Key Laboratory for Microbes and Functional Genomics,Nanjing 210023,China
Keywords:
chromosome-based; recombineering; TEV; protein expression
PACS:
Q819
DOI:
-
Abstract:
Due to its high proteinase cleavage activity,specificity and effective in a wide range of conditions,Tobacco etch virus protease(TEV)has many applications,ranging from protein isolation to proteomics study.Currently,TEV is produced by plasmid-based overexpression in Escherichia coli BL21(DE3).Yet,the method has inherent disadvantages,for example,it needs antibiotic to maintain the plasmid which may bring impurities during the protein purification; and non-homogeneity of the strain population which may reduce the yield.Herein,we report the recombineering mediated integration of MBP fused TEV gene under the strong T7 promoter into E.coli BL21(DE3)chromosome.Intracellular digestion of chromosomal based overexpression of MBP-TEV released TEV which was subsequently isolated though Ni-NTA affinity purification,the yield of TEV was up to 4.2 mg/L.Purified TEV shows fine protease activity.The engineered strain has the potential to be used for large scale TEV purification,the established recombineering method can be a platform for the genome engineering and heterologus gene expression in E.coli BL21(DE3).

References:

1] Waugh D S.Making the most of affinity tags[J].Trends Biotechnol,2005,23(6):316-320.
[2]Kapust R B,Tozser J,Fox J D,et al.Tobacco etch virus protease:mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency[J].Protein Eng,2001,14(12):993-1 000.
[3]Babu M,Krogan N J,Awrey D E,et al.Systematic characterization of the protein interaction network and protein complexes in Saccharomyces cerevisiae using tandem affinity purification and mass spectrometry[J].Methods Mol Biol,2009,548:187-207.
[4]Chen I,Dorr B M,Liu D R.A general strategy for the evolution of bond-forming enzymes using yeast display[J].Proc Natl Acad Sci USA,2011,108(28):113 399-113 404.
[5]Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proc Natl Acad Sci USA,2000,97(12):6 640-6 645.
[6]Isaacs F J,Carr P A,Wang H H,et al.Precise manipulation of chromosomes in vivo enables genome-wide codon replacement[J].Science,2011,333(6 040):348-353.
[7]Wang H H,Kim H,Cong L,et al.Genome-scale promoter engineering by coselection MAGE[J].Nat Methods,2012,9(6):591-593.
[8]Kuhlman T E,Cox E C.Site-specific chromosomal integration of large synthetic constructs[J].Nucleic Acids Res,2010,38(6):e92.
[9]张飞飞,王瑞青,朱宇鹏,等.大肠杆菌系列融合表达载体的构建[J].南京师大学报:自然科学版,2013,36(3):97-102.
[10]Sambrook J,Fritsch E F,Maniatis T.Molecular Cloning:A Laboratory Manual[M].2nd ed.New York:Cold Spring Harbor Laboratory Press,1989.
[11]Jere P S,Richard L J.Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate[J].Meth Enzymol,1972,28:54-63.
[12]Takahashi S,Ohsawa T,Miura R,et al.Purification of high molecular weight(HMW)renin from porcine kidney and direct evidence that the HMW renin is a complex of renin with renin binding protein(RnBP)[J].J Biochem,1983,93(1):265-274.

Memo

Memo:
-
Last Update: 2014-09-30