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

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

Soluble Expression of Recombinant Human Bone Morphogenetic Protein-7 (rhBMP-7) in Escherichia coli Using SUMO Fusion System: Soluble expression of rhBMP-7 in E. coli using SUMO fusion

BMPs belong to transforming growth factor β superfamilies, which their principal role is inducing bone and cartilage formation at heterotopic and orthotopic sites. Since the formation of inclusion bodies is the main limitation of producing these proteins in Escherichia coli, in this study, the small ubiquitin-like modifiers (SUMO) fusion system was employed to improve solubility and expression of recombinant human BMP-7 (rhBMP-7) in E. coli. The SUMO fusion system has the ability to enhance protein expression, reduce target protein proteolytic degradation, and increase protein folding and solubility. In the current study, the SUMO protein gene was fused to the N-terminus of the BMP-7 gene, and cloned in the pET-28a vector. After purification of the expressed SUMO-BMP-7 protein by Ni-NTA chromatography, SUMO was removed from the BMP-7 protein using SUMO protease. In the second step of purification using Ni-NTA chromatography, the cleaved BMP-7 protein was purified and then identified by Western blot analysis. The results of the current study demonstrated that the SUMO fusion system is able to increase the soluble form of rhBMP-7. Furthermore, rhBMP-7 can be purified by a two-step purification strategy including: 1) purification of SUMO-BMP-7 and 2) purification of rBMP-7 after cleavage using Ni-NTA chromatography. Altogether, this research has provided a feasible approach for large-scale production of soluble rhBMP-7, to facilitate its further medical development. HIGHLIGHTS SUMO is a well characterized family of ubiquitin-like molecules. SUMO fusion led to increased expression and solubility of BMP-7. BMP-7 is involved in the process of bone formation

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

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

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios

Western blot analyses of purified mAb in triplicate stable cell pools transfected with vectors bearing F2A-mediated expression system.

<p>Light chains with various molecular weights (25, 28 & 30 kDa) were detected. Triplicates of each stable pool were shown with 1, 2 and 3. (A) Lane 1; CHEF-F2A (2), lane 2; CHEF-F2A (3), lane 3; protein molecular ladder. (B) Lane 1; protein molecular ladder, lane 2; CMV-F2A (1), lane 3; CMV-F2A (3).</p

Analysis of the stability of antibody expression over time by stable cell pools transfected with two vectors containing F2A sequences.

<p>Both stable cells were cultivated for 18 weeks upon removal of puromycin as a selection marker. Every 2 weeks, antibody expression was monitored and measured by ELISA. The other pools exhibited the same expression pattern; the data from one of them was represented.</p

SDS-PAGE and Western blot analysis of purified mAb in stable cell pools transfected with four bicistronic vectors.

<p>Supernatants of three different cell pools were purified using protein-A. Purified samples were analyzed with SDS-PAGE and western blot under reducing and non-reducing condition. Commercial IgG1 was used as a positive control. (A) SDS-PAGE profile of purified samples in reduced state, lane 1; Protein molecular ladder, 2; CHEF-F2A, 3; CMV-F2A, 4; CHEF-IRES, 5; CMV-IRES, 6; positive control. (B) SDS-PAGE profile of purified samples in non- reduced state, lane 1; CMV-IRES, 2; CHEF-IRES, 3; CMV-F2A, 4; CHEF-F2A, 5; protein molecular ladder. (C) Western blot analysis of samples under reducing condition, lane 1; protein molecular ladder, 2; CHEF-F2A, 3; CMV-F2A, 4; CHEF-IRES, 5; CMV-IRES, 6; positive control. (D) Western blot analysis of purified samples in non- reduced state, lane 1; CMV-IRES, 2; CHEF-IRES, 3; CMV-F2A, 4; CHEF-F2A, 5; protein molecular ladder.</p