mozhgan raigani

CRISPR is a powerful technology and the CRISPR-Cas9 system has provided a new tool for simple, precise, selective and easy to use genetic engineering of any different DNA targets of the genome. This ability is essential for understanding gene function, engineering cell behaviors, and developing therapies. The type II CRISPR-Cas9 system is the most commonly used for genome editing. This ribonucleoprotein complex consists of a DNA endonuclease (Cas9) and two RNAs, CRISPR RNA (crRNA) and transacting RNA (tracrRNA). Subsequently, the discovery of nuclease-dead Cas molecules (dCas9) provided a platform for precise control of genome function without gene editing. In this review article, the advances of CRISPR/dCas9 technique and its applications in the field of genome engineering and gene expression, as well as the improvement of its methods, have been discussed.

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.

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).

Increased efficiency was achieved by EnBase® compared to Luria–Bertani 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.

Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

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.  

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.

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.

The relation between key enzymes in regulation of folate metabolism and male infertility is the subjectof numerous studies. We aimed to determine whether 5, 10-methylenetetrahydrofolate reductase (MTHFR) C677Tand methionine synthase reductase (MTRR) A66G genotypes are associated with male infertility in Iranian men andto evaluate its effect on seminal levels of folate and vitamin B12.

In this retrospective study, semen and peripheral blood samples were collected from 254men 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.

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 normozospermicmen (522.6 ± 388.1 pg/ml) compared to OAT men (412.9 ± 303.6 pg/ml, P=0.058).

The MTHFR C677T and MTRR A66G have no effect on the concentrations of seminal folate and vitaminB12. The present study showed that two SNPs of MTRR A66G and MTHFR C677T cannot be seen as a risk factor formale factor subfertility.

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.
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.
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.
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.
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.

Culture media enrichment through the addition of protein hydrolysates is beneficial for achieving higher protein expression.
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.
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.
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.