Computational analysis of fusion protein of anti-HER2 scFv and alpha luffin: A new immunotoxin protein for HER2 positive cancers

Abstract The present study deals with the computational design and analysis of a novel fusion protein based on a single chain variable fragment that binds to the extracellular domain of human epidermal growth factor receptor 2 (HER2) in breast cancer cells. Alpha luffin, a small ribosome inactivating protein (RIP), was attached to the anti-HER2 antibody fragment. I-TASSER modeling provided the full-length structure of the fusion protein. Molecular docking evaluated the molecular interactions of the complementarity-determining regions of designed fusion protein to HER2. Energy minimization and molecular dynamics simulations were conducted to refine the complexes. RMSD plot revealed reasonable stability of the fusion protein during the simulation. The free binding energy profile of complexes affirmed a favorable binding affinity of proteins in complex with HER2 using molecular mechanics Poisson-Boltzmann surface area (G-MMPBSA) algorithm. In general, this approach looks promising in the development of new fusion proteins in terms of immunotoxins with appropriate cytotoxicity.

Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.