title: Co-expression of disulfide oxidoreductases DsbA/DsbC markedly enhanced soluble and functional expression of reteplase in Escherichia coli.
abstract: Reteplase is the third generation of thrombolytic medicine and has many advantages over commercial t-PA. However, over-expressing recombinant reteplase in E. coli always accumulates as inclusion bodies due to nine pairs of disulfide bonds formation that is the main obstacle for correct folding. In this paper, in order to enhance soluble expression of recombinant reteplase in E. coli, DsbA/DsbC foldases were used to introduce disulfide bonds into the reduced polypeptide chain and catalyze their isomerization to the native disulfide linkage during the folding process. Firstly multiple E. coli protein expression systems, i.e. DsbA, DsbC and DsbA/DsbC co-expression were constructed. Subsequently, IPTG and l-arabinose were added to induce expression of foldases and reteplase accordingly, and experimental parameters such as culture temperature and inducer concentration were optimized. As a result, the co-expression system markedly enhanced soluble expression of recombinant reteplase, and up to 60% of reteplase achieved soluble expression especially for the DsbC co-expression system. The fibrin plate method for active reteplase quantification showed that ∼70 mg soluble reteplase per liter fermentation broth was obtained with 2.35 × 105 IU/mg thrombolytic activity. Finally, fluorescence spectra indicated that the structural conformation of soluble reteplase was identical to its native state. The soluble expression of recombinant reteplase in E. coli was accomplished by co-expression with DsbA/DsbC, which contributes to further research in clinical application and folding mechanism, and provides guidance for production of other proteins with disulfide bonds.
authors.forename: Xiao-Fa
authors.initials: XF
authors.lastname: Zhuo
authors.forename: Yi-Ying
authors.initials: YY
authors.lastname: Zhang
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Guan
authors.forename: Shan-Jing
authors.initials: SJ
authors.lastname: Yao
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism; D010957:Plasmids; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005337:Fibrin; C087896:reteplase; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli; C526649:dsbC protein, E coli