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Enhancement of cytoplasmic solubility of heparinase I from Flavobacterium heparinum in Escherichia coli by adding charged tag and co-expression of molecular chaperonin from hyperthermophilic archaea.
Guo Yu 1,Li Huichao 2,Zhang Hao 2,Fujiwara Shinsuke 2,Gao Le 2 *
1. School of Life Science and Technology, Changchun University of Science and Technology, Weixing Road 7186, Changchun, 130022, China; School of Life Science and Technology, Changchun University of Science and Technology, Weixing Road 7186, Changchun, 130022, China;School of Life Science and Technology, Changchun University of Science and Technology, Weixing Road 7186, Changchun, 130022, China; Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan; School of Life Science and Technology, Changchun University of Science and Technology, Weixing Road 7186, Changchun, 130022, China
2.
*Correspondence author
#Submitted by
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Funding: foundation of Jilin Province Science and Technology Department （No.No. 20200801062GH）
Opened online:22 February 2021
Accepted by: none
Citation: Guo Yu,Li Huichao,Zhang Hao.Enhancement of cytoplasmic solubility of heparinase I from Flavobacterium heparinum in Escherichia coli by adding charged tag and co-expression of molecular chaperonin from hyperthermophilic archaea.[OL]. [22 February 2021] http://en.paper.edu.cn/en_releasepaper/content/4753657
 
 
Heparinase is important in the heparin production and application. The most studied heparinase I is HepA which is secreted by Flavobacterium heparinum. Since HepA was hard to obtain as a soluble form in Escherichia coli, many works were tried to increase cytoplasmic solubility of HepA by adding various protein tags to the HepA terminus. Here we designed a positively charged hexapeptide tag (RGRRGG) which was expected to have enhanced affinity to a chaperonin CpkA from hyperthermophilic archaeaon Thermococcus kodakarensis. HepA was first fused with RGRRGG and expressed by plasmid pET21a. Effect of tag addition on cytoplasmic soluble amount of HepA in E. coli were examined, showing that the amount of the tag fused HepA(HepA\') was boosted in comparison with tag-less original one. HepA\' was next co-expressed with a CpkA by another compatible plasmid pACYCDuet-1. By CpkA co-expression, insoluble amount of HepA\' was decreased and soluble amount of HepA\' was increased and reached to 40% of total expressed amount of HepA\'. The specific activity (126 IU/mg) was also comparable to the native HepA (130 IU/mg). The present data indicated that tag-charged HepA was efficiently obtained as an active form by co-expressing CpkA. (10 Points, Times New Roman)
Keywords:Biochemistry & Molecular Biology; Heparinase I; chaperonin; polyionic peptide tag; soluble expression
 
 
 

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