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Published on: August 3, 2017
Comparison of truncated human angiotensin-converting enzyme 2 (hACE2) expression in pET28a(+) versus pET-SUMO vector
Dora Domnica Baciu1, Sorin Dinu1, Bianca Palade1
1Molecular Biology and Proteomics Department, Cantacuzino National Medical-Military Institute for Research and Development, Bucharest, Romania.
Purpose:
Truncated human angiotensin-converting enzyme 2 (hACE2) expression rises a great scientific interest, considering its possible therapeutic and diagnostic applications. A promising research direction is the therapeutic use of smaller hACE2 versions with high binding affinity as decoy receptors for S1 glycoprotein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Another possible application is the use of these truncated versions for the functionalization of appropriate nanomaterials for constructing novel biosensors with a rapid and sensitive response for coronavirus disease 2019 (COVID-19) detection. The present study aimed to find a suitable system for high yield expression of different versions of truncated hACE2.
Materials And Methods:
The encoding DNA for the hACE2 fragments (7-507 aa, 16-128 aa, and 30-357 aa) was obtained by PCR amplification using as template pcDNA3.1-hACE2 plasmid and further cloned into pET28a(+) and pET-SUMO vectors. The positive clones were selected and the correct DNA insertion was confirmed through gene sequencing. The truncated hACE2 proteins were further expressed in two E. coli strains, Rosetta(DE3) and BL21(DE3).
Results:
For all three truncated hACE2 mini proteins, pET28a(+) does not lead to protein expression, regardless of the bacterial strain. The situation changes with the use of the pET-SUMO expression system when all hACE2 fragments are expressed, but with higher efficiency in E. coli BL21(DE3) than E. coli Rosetta.
Conclusion:
In the present study, we showed that different versions of recombinant hACE2 are successfully expressed in E. coli BL21(DE3) by using pET-SUMO expression system.
