Comparison of truncated human angiotensin-converting enzyme 2 (hACE2) expression in pET28a(+) versus pET-SUMO vector and two Escherichia coli strains.

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.

Generation of a 3D melanoma model and visualization of doxorubicin uptake by fluorescence imaging.

Melanoma is the most dangerous type of skin cancer and is responsible for 75% of deaths from skin cancers. For an accurate evaluation of potential treatment efficacy, it is important to use study models as close as possible to the in vivo conditions. A 3D model consisting of B16F10 spheroids was developed using liquid overlay technique on plates coated with 1% agarose, in the presence of 1% methylcellulose and L929-conditioned medium. The model is suitable and can be further used for more complex in vitro drug testing than the classical 2D approach. For exemplification, the behavior of a well-known cytostatic, doxorubicin (DOX), was evaluated in spheroids as compared to classical 2D culture conditions. Fluorescence imaging was used to visualize DOX uptake by B16F10 spheroids at different periods of time. The results showed that a much higher DOX concentration is necessary to produce similar effects compared with the monolayer. The fluorescence images revealed that at least 4 h of stimulation is needed for a sufficient DOX uptake. The 3D model developed in this study was suitable to investigate drug penetration in time. Our findings may explain the decrease of the doxorubicin therapeutical effect, suggesting the need of maintaining the drug concentration at the tumoral place for at least 2 h upon administration. Similar or more advanced studies can lead to a better understanding of drug delivery kinetics and distribution upon administration, conducing toward a better performance in designing suitable delivery systems for obtaining the optimum dose-response effect.