sFLT01 chimeric protein sequence suggestion, tertiary structure prediction, expression of construct in host cells and In vitro anti angiogenic functional analysis

Session Details

Session Title: Free Paper Session 18: New Drug & Treatment Technology

Session Date/Time: Saturday 09/09/2017 | 14:30-16:00

Paper Time: 15:00

Venue: Room 120

First Author: : D.Samiee IRAN

Co Author(s): :    F. Zakeri   Z. Soheili   M. Sadeghi   S. Samiee   E. Ranaei Pirmardan   H. Ahmadieh              

Abstract Details

Purpose:

sFLT01 is an engineered chimeric secretory protein with the inhibitory effect on VEGF and PLGF which are expressed aberrantly in AMD ocular disease. The aim of this study was its protein sequence suggestion, tertiary structure prediction, expression of its cDNA in host cells and evaluation of its inhibitory function by tube formation assay.

Setting:

National Institute of Genetic Engineering and Biotechnology

Methods:

To determine the sequence, we achieved to the FLT1 second domain amino acid sequence and thereafter corresponding nucleotide sequence was determined. Using EMBOSS Transeq tool of EBI database and NCBI BLAST tool our proposed sequence got validated. Finally, modeler servers and soft wares applied for the proposed sequence structure validation. We determined tertiary structure of the sequence in its chimeric form and compared it with constitutive components in natural forms. To predict tertiary structure, we used modeler servers such as I-TASSER, Swiss model, phyre2 and psipred. The prediction results analyzed with PDBviewer and PyMOL soft wares. hRPE cells was transfected by the expression construct and sFLT01 secretion was detected in culture medium and in cell lysates by western blotting. We demonstrated anti angiogenic function of conditioned media by in vitro angiogenesis method.

Results:

Software analysis demonstrated that, proposed chimeric construct was in an excellent accordance with its natural counterpart based on its tertiary structure and preserved its natural folding exactly. Western blotting data showed sFLT01 secretion into the culture medium of hRPE transfected cells. In Vitro angiogenesis assay results showed decreased potential of tube formation in conditioned medium of RPE cultures that had been treated by constructs expressing SFLT01.

Conclusions:

According to predicted tertiary structure, our suggested sequence for sFLT01 was an excellent counterpart for its natural components. It was also expressed, secreted, and inhibited tube formation of HUVEC cells successfully.

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