Co-expression Of Disulfide Oxidoreductases DsbA/DsbC Markedly Enhanced Soluble And Functional Expression Of Recombinant Reteplase In Escherichia Coli

Reteplase (rt-PA), the third generation of thrombolytics for the treatment of ischemic stroke, is a deletion mutant of wild tissue-type plasminogen activator (t-PA) through genetic engineering. Compared with the second generation of thrombolytics (tissue type plasminogen activator), certain structural changes bring about rt-PA some advantages such as prolonged half-life, increased fibrinolytic potential, more rapid and complete coronary patency, and little adverse effects etc, all of which endow reteplase with a good application prospect. 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 reteplase gene sequence was optimized according to the codon preference of E. coli. And then multiple E. coil protein expression systems, i.e. DsbA, DsbC and DsbA/DsbC co-expression were constructed by cloning reteplase gene and DsbA/DsbC gene into pBAD/HisA and pACYCDuet-1plasmids respectively. Agarose gel electrophoresis was used to verify the successful construction of co-expression systems.Subsequently, the influences of some experimental parameters such as induction method and time, culture temperature and inducer concentration on reteplase soluble expression were studied and the co-expression system markedly enhanced soluble expression of recombinant reteplase. Host cells were cultured at37℃、200rpm until OD600reached about0.6, and then0.1mM IPTG was added to induce DsbA/DsbC expression. After further culturing1h at25℃、160rpm,0.5g/L L-arabinose was added to induce reteplase expression for10h. As a result, up to60%of reteplase achieved soluble expression especially for the DsbC co-expression system, and-70mg soluble reteplase per liter fermentation broth was obtained.Finally, soluble reteplase was purified by Ni-NTA metal-affinity chromatography. Fluorescence spectra indicated that the structural conformation of soluble reteplase was identical to its native state. The fibrin plate method was used to assay the thrombolytic activity of purified reteplase, and the thrombolytic activity was2.35×105IU/mg. The two models for disulfide bonds isomerization catalyzed by DsbC were studied and the isomerization model was discussed in detail.The soluble expression of recombinant reteplase in E. coil 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.