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.

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results: : Increased efficiency was achieved by EnBase® compared to Luria­Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 µg/mL. Conclusion: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells.

Background: Bispecific antibodies represent an important class of monoclonal antibodies (mAbs), with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti-CD3 × CD19 bispecific antibody (bsAb) is a bispecific T-cell engager currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods: Woodchuck hepatitis virus post-transcriptional regulation (WPRE) sequence was incorporated at the 3' end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion: Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in human embryonic kidney-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody.

Targeted integration into pseudo attP sites of CHO cells using CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and fluorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.

A Association of MTHFR C677T and MTRR A66G Gene Polymorphisms with Iranian Male Infertility and Its Effect on Seminal Folate and Vitamin B12.

BACKGROUND: The relation between key enzymes in regulation of folate metabolism and male infertility is the subject of numerous studies. We aimed to determine whether 5, 10-methylenetetrahydrofolate reductase (MTHFR) C677T and methionine synthase reductase (MTRR) A66G genotypes are associated with male infertility in Iranian men and to evaluate its effect on seminal levels of folate and vitamin B12. MATERIALS AND METHODS: In this randomized clinical trail study, semen and peripheral blood samples were collected from 254 men with oligoasthenoteratozoospermia (OAT) and 77 normozoospermic men who attended Avicenna infertility clinic. Single nucleotide polymorphism (SNP) analysis was carried out in genomic DNA by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for MTHFR C677T and MTRR A66G gene polymorphisms. RESULTS: In MTHFR C677T, our founding showed that T carrier was conversely lower in OAT than normozoospermic men (χ2-test=7.245, P=0.02) whereas in MTRR A66G, A and G carrier showed no significant difference between the two groups (χ2-test=1.079, P=0.53). The concentration of seminal folate was not different between normozoospermic (18.83 ± 17.1 ng/ ml) and OAT (16.96 ± 14.2 ng/ml) men (P=0.47). The concentration of vitamin B12 was slightly higher in normozospermic men (522.6 ± 388.1 pg/ml) compared to OAT men (412.9 ± 303.6 pg/ml, P=0.058). CONCLUSION: The MTHFR C677T and MTRR A66G have no effect on the concentrations of seminal folate and vitamin B12. The present study showed that two SNPs of MTRR A66G and MTHFR C677T cannot be seen as a risk factor for male factor subfertility.

Preparation and investigation of indirubin-loaded SLN nanoparticles and their anti-cancer effects on human glioblastoma U87MG cells.

Indirubin, an ingredient in traditional Chinese medicine, is considered as an anti-cancer agent. However, due to its hydrophobic nature, clinical efficiency has been limited. Drug delivery via nanotechnology techniques open new windows toward treatment of cancerous patients. Glioblastoma multiforme (GBM) is the most severe and common type of brain primary tumors. Of common problems in targeting therapies of glioblastoma is the availability of drug in tumoric tissues. In this study, Indirubin loaded solid lipid nanoparticles were prepared and their therapeutic potentials and antitumoric effects were assessed on GBM cell line (U87MG). The SLNs were prepared with Cetyl palmitate and Polysorbat 80 via high-pressure homogenization (HPH) methods in hot mode. Then, properties of SLNs including size, zeta potential, drug encapsulation efficacy (EE %) and drug loading were characterized. SLNs morphology and size were observed using SEM and TEM. The crystalinity of formulation was determined by different scattering calorimetry (DSC). The amount of drug release and antitumor efficiency were evaluated at both normal brain pH of 7.2 and tumoric pH of 6.8. The prapared SLNs had mean size of 130 nm, zeta potential of -16 mV and EE of 99.73%. The results of DSC showed proper encapsulation of drug into SLNs. Drug release assessment in both pH displayed sustain release property. The result of MTT test exhibited a remarkable increment in antitumor activity of Indirubin loaded SLN in comparison with free form of drug and blank SLN on multiform GB. This study indicated that Indirubin loaded SLNs could act as a useful anticancer drugs.

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein.

BACKGROUND: The increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins. OBJECTIVES: Here, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined. MATERIALS AND METHODS: The encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column. RESULTS: In fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C. CONCLUSIONS: Our founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

The Potential Use of Peptides in Cancer Treatment.

Conventional chemotherapeutic drugs have significant limitations. For example, tumors may develop resistance, cancers may relapse after treatment, and the drugs may induce secondary malignancies in the treatment of metastatic cancer. There is still a great need for drugs that are able to destroy cancer cells selectively, that is, to effectively treat slow-growing and dormant cells without being affected by chemoresistance mechanisms. A growing number of studies indicate that peptides may be beneficial for drug discovery and development. Peptides offer minimal immunogenicity, excellent tissue penetrability, low-cost manufacturability, and ease of modification for enhancing in vivo stability and biological activity, properties which make them ideal candidates for cancer treatment. This review highlights recent advances in and future prospects for the application of peptides as therapeutic agents for cancer therapy. We discuss the application of peptides in cancer therapy, alone and in combination with other peptides or small-molecule chemotherapeutic drugs, for use in targeted cancer therapy. Furthermore, we consider the use of peptides as a carrier for targeted molecular imaging in the diagnosis and follow-up treatment of cancer. This account also reviews the challenges of using peptide drugs and ways to overcome these limitations. The results obtained in studies presented in this paper indicate that peptides are promising candidates for targeted cancer therapy.

Cloning and soluble expression of mature α-luffin from Luffa cylindrica in E. coli using SUMO fusion protein.

α-Lufin, found in Luaf cylindrica seeds, is a type I ribosome inactivating proteins. Cytotoxic effects make it an appropriate candidate for the construction of immunotoxins and conjugates. Because of limited natural resources, recombinant technology is the best approach to achieve large-scale production of plant-based proteins. In the present study, α-lufin protein was expressed in E. coli and the effects of different temperature conditions, SUMO fusion tag, and cultivation strategies on total expression and solubility were investigated. Protein expression was evaluated at different intervals (0, 4, 6, 8, 24 h) postinduction. Our results showed that EnBase had higher eficiency than LB, and maximum solubility and total protein expression were achieved 24 h after induction at 30 °C and 25 °C, respectively. It was shown that SUMO tag is an effective strategy to improve protein solubility.

Rapid characterization of the CHO platform cell line and identification of pseudo attP sites for PhiC31 integrase.

The Chinese Hamster Ovary (CHO) cell lines, applicable to post-translational modifications, are preferred systems for biopharmaceutical protein production. In this study, by using the Jump-In™ TI™ technology which employs PhiC31 and R4 bacteriophage recombinases, a platform CHO-K1 cell line containing a R4-attP site was generated. Here, a combination of Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR) and semi-random, two-step PCR (ST-PCR), was performed to feature the platform cell clones. Our results show that QF-PCR and ST-PCR, can be utilized for efficient and accelerated cell line characterization. By applying these approaches, we were able to accurately identify the copy number of integrated R4-attP sites and the genomic position of recombination of many clones. Three novel PhiC31 pseudo-attP sites were identified on chromosomes 1, 3 and 6, and their genomic features were analyzed. The characterized platform cell lines are stable, and because of single-copy, site-specific R4 recognition attP site, the cell lines could be retargeted for recombinant protein production and drug discovery applications.

Proteomics Profiling of Chimeric-Truncated Tissue Plasminogen activator Producing- Chinese Hamster Ovary Cells Cultivated in a Chemically Defined Medium Supplemented with Protein Hydrolysates

Background: Culture media enrichment through the addition of protein hydrolysates is beneficial for achieving higher protein expression. Methods: In this study, designing the optimum mixture of four soy and casein-derived hydrolysates was successfully performed by design of experiment and specific productivity increased in all predicted combinations. Protein profile of recombinant CHO (rCHO) cells producing tissue plasminogen activator in a serum-free medium (SFM) supplemented with designed hydrolysate additives was compared to that of rCHO cells cultivated in SFM. Results: Identification of differentially expressed proteins using two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF revealed the role of energy metabolism related proteins and importance of prevention of oxidative stress by this special media enrichment strategy. Up-regulation of mitochondrial enzymes, pyruvate dehydrogenase E1 and Peroxiredoxin-III, as well as other proteins involved in metabolic pathways, and uridine monophosphate/cytidine monophosphate kinase indicated higher metabolic activity. Furthermore, along with antioxidant effect of peptones, proteins with antioxidant function such as ferritin and peroxiredoxin-III were up-regulated. Conclusion: Understanding molecular mechanisms involved in enhancement of protein expression can provide new approaches for efficiently engineering rCHO cell. These results support the competence of proteomics studies in finding new insights to biochemical pathways for a knowledge-based optimization of media compositions.

Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats.

Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC0→∞) increased. The elimination half-life (t1/2) of mt-PA was in the range of 19.1-26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3-31.8 min in three doses, which was 4-5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies.

Nonsense mediated decay of VWF mRNA subsequent to c.7674-7675insC mutation in type3 VWD patients.

Von Willebrand disease (VWD), the most common genetic bleeding disorder, is caused by defects in Von Willebrand factor (VWF). Quantitative deficiencies of the protein lead to either VWD type3, the severe form of the disease or VWD type1 with milder clinical manifestation. Null alleles are the most common mutations in VWF gene causing type3. However, some of these mutations are not translated into the protein and are selectively degraded at mRNA level by nonsense-mediated decay (NMD) pathway. Here, we have studied a large VWD type3 pedigree with a premature termination codon (PTC) causing insertion mutation (c.7674-7675insC) in VWF exon 45. We further investigated the impact of the mutation on the VWF mRNA expression using a quantitative Real-time PCR assay and cDNA sequencing. The relative expression of the gene was significantly decreased in the patients' platelets (Mean ratio=0.03 (0.01-0.05), p=0.001) compared to their normal relatives. The heterozygote carriers of the mutation had lower than normal VWF mRNA levels (Mean ratio=0.62 (0.29-0.91), p=0.006). Direct sequencing of exon 45 on the platelet-derived cDNA in the carriers revealed only the wild-type allele confirming the decay of the mutation carrying allele. In conclusion, quantitative analysis of VWF gene expression showed that c.7674-7675insC mutation in VWF gene resulted in degradation of VWF mRNA via NMD. This pathway might play an important role in the pathogenesis of VWD characterized by quantitative deficiency of VWF due to reduced mRNA levels.