Improved production of holotoxin Stx2 with biological activities by using a single-promoter vector and an auto-induction expression system.
The entire stx2 region from Escherichia coli O157:H7, containing two open reading frames (stx2a and stx2b), was cloned into pET-32a with a single promoter, and transformed into E. coli BL21 (DE3) pLysS. We used two methods of IPTG induction using LB medium and auto-induction using ZYM-5052 medium to express recombinant Shiga toxin 2 (rStx2). rStx2 was expressed in the E. coli periplasm in a completely soluble, biologically active form. The final yield of purified rStx2 from each liter of culture in LB medium and ZYM-5052 medium was approximately 2.3mg and 3.5mg, respectively. The highest amount of rStx2 accounted for 27.8% of total bacteria protein in ZYM-5052 medium. rStx2A and rStx2B isolated from rStx2 by electroelution were, respectively, identified by N-terminal protein sequencing. Signal peptides with the sequence MKCILFKWVLCLLLGFSSVSYS and MKKMFMAVLFALASVNAMA were identified at the N terminus of rStx2A and rStx2B, respectively. Our rStx2 possessed Vero cell CD(50) value about 500pg and LD(50) value approximately 6ng. rStx2 can be substitute for natural toxin Stx2, which can be used for animal models, drug screening, vaccine research, and so on.
Tu
Wei
W
State Key Laboratory of Pathogens and Biosecurity, Institute of Microbiology and Epidemiology, No. 20 Dongdajie, Fengtai District, Beijing 100071, China.
Cai
Kun
K
Gao
Xiang
X
Xiao
Le
L
Chen
Rongchang
R
Shi
Jing
J
Liu
Hao
H
Hou
Xiaojun
X
Wang
Qin
Q
Wang
Hui
H
D000818:Animals; D002470:Cell Survival; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020539:Sequence Analysis, Protein; D022641:Shiga Toxin 2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014709:Vero Cells
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D022641:Shiga Toxin 2