title: Construction, expression and refolding of a bifunctional fusion protein consisting of C-terminal 12-residue of hirudin-PA and reteplase.
abstract: To obtain a bifunctional protein simultaneously showing bioactivity of anticoagulant and fibrinolytic for use in the treatment of thrombotic diseases, we constructed a fusion protein (HV12p-rPA) containing C-terminal 12-residue of hirudin-PA (HV12p) and reteplase (rPA). The fusion protein, in which HV12p was linked to rPA via Gly-Gly-Gly, was successfully expressed in an inactive form of inclusion bodies in Escherichia coli. HV12p-rPA was identified by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The expression level of HV12p-rPA was optimized by an orthogonal method and finally enhanced from 12 % to approximate 30 %. We also deeply investigated the condition of renaturation of HV12p-rPA, and the inactive protein was partly renatured through various conditions. The refolding efficacy of HV12p-rPA estimated by the recovery of fibrinolytic activity varied from 0.03 % to 16.6 % and the anticoagulant activity fluctuated in the range from 41 to 2,297 ATU/mg. Bioassays indicated that the resulted fusion protein, as expected, exhibited both fibrinolytic and anticoagulant activities. These works laid a foundation for further characterization of HV12p-rPA.
authors.affiliation: Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Gao
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.forename: Lingling
authors.initials: L
authors.lastname: Li
authors.forename: Lan
authors.initials: L
authors.lastname: Liang
authors.forename: Xuan
authors.initials: X
authors.lastname: Deng
authors.forename: Wutong
authors.initials: W
authors.lastname: Wu
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
authors.forename: Rong
authors.initials: R
authors.lastname: Yu
mesh_terms: D020816:Amino Acid Motifs; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D006629:Hirudins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C067832:hirudin PA; C087896:reteplase; D010959:Tissue Plasminogen Activator