Journal of Applied Biotechnology Reports
Volume:10 Issue: 1, Winter 2023

Escherichia coli is one of the most commonly used organisms for producing recombinant protein. The recognized cell genome and well-known cell factory of the bacteria make it an ideal heterologous system of preference for the production of recombinant proteins. Over recent years, this cell system has been modified to improve the production of therapeutic proteins, and several methodologies are now available. One of the scientists' most preferred strategies is maintaining cell survival by optimizing the culture conditions. This review summarises some experiments related to those culture conditions and discusses how they affect recombinant protein expression in E. coli.

Vaccination is the most effective method to prevent dangerous infectious diseases and save lives. The expansion of human communication, the rapid spread of emerging infections worldwide, and the creation of dangerous pandemics like COVID-19 is worrying. On the other hand, with the emergence of new technologies such as genetic engineering of microorganisms, genome editing, and synthetic biology, the possibility of abusing these tools for illegal use is the next concern. In this situation, the need for rapid vaccination technologies and programs was given special importance. Recently, new vaccine platforms such as viral vector and mRNA vaccines have shown great promise that they can be used to prepare and protect human lives against dangerous infections. One of the most important factors for vaccination is the rapid development and approval of vaccines. In this review, we have given a perspective view of new vaccine technologies to rapidly develop vaccines to combat emerging infections and the biodefence against biological criminals.

The phage display method is a technology that enables the expression of exogenous polypeptides on the surface of bacteriophage particles. Phage titration and ELISA are applied to measure helper phage particles or polypeptide bearing phages and also evaluation the interaction between polypeptide bearing phages and coated antigens, respectively. Although several procedures have been introduced to perform phage titration and ELISA, they face some limitations, such as being time-consuming, expensive, and low reproducibility. We developed a new system called EnzyPha by engineering the M13KO7 expressing Secreted Acid Phosphatase of Mycobacterium tuberculosis (SapM enzyme) on its pIX protein for applying in colorimetric phage titration and ELISA methods. To evaluate the idea, colorimetric phage titration and ELISA were performed and compared to the traditional methods. SapM enzyme was expressed on the pIX protein of M13KO7 properly. The colorimetric phage titration and phage ELISA showed better and comparable results against the traditional approaches. The results showed that the proposed model would titrate phages more sensitively than the plating titration method through a shorter timeframe. Moreover, it could be a better alternative to the routine phage ELISA due to time-saving, cost-effectiveness, and higher sensitivity.

Endophytic fungi are good sources of bioactive compounds that are exclusive to their hosts. Eichhornia crassipes plant produces different bioactive compounds. The aim of the present study is to isolate and identify endophytic fungi that reside in Eichhornia crassipes tissues, and to evaluate the biological activities of their extracts. Endophytic fungal spp. were isolated from leaves and petioles of Eichhornia crassipes, identified and then extracted. The ethyl acetate extracts were tested against bacteria, fungi, hepatitis B virus and Schistosoma mansoni cercariae. The chemical composition of these extracts was determined by Gas chromatography-mass spectrometry (GC- MS/MS) analysis. We found that four fungal spp. were dominant in Eichhornia crassipes; they were molecularly identified as Aspergillus flavus OM758315, Aspergillus fumigatus OM688980, Aspergillus welwitschiae OM758326 and Corynascus sepedonium OM688206, with A. flavus as the most frequent. The ethyl acetate extract of the four fungal spp. showed pronounced antimicrobial effects, whereas the highest antiviral effect on hepatitis B virus was that of A. flavus followed by A. fumigatus extracts. All the tested extracts were cercaricidal to Schistosoma mansoni cercariae, where A. flavus was the most effective. GC- MS/MS analysis indicated the presence of various bioactive compounds. Aspergillus flavus, Aspergillus fumigatus, Aspergillus welwitschiae and Corynascus sepedonium as endophytes of Eichhornia crassipes showed promising antimicrobial, antiviral and cercaricidal properties.

Keratocytes are the major components of the human corneal stromal cell. Cell therapy by keratocytes can be used in some corneal diseases. Because keratocytes are mitotically quiescent; therefore, the cultivation of these cells is associated with challenges. The present study aimed to isolate, culture, and validate keratocyte cells from discarded corneal tissue based on optimizing some cultivation conditions. In this experimental study, keratocytes were isolated from discarded corneal tissue. Different culture medium composition such as amniotic membrane extract, time, and the role of coating scaffolds was evaluated. Real-time PCR of specific genes were used to confirm the primary keratocyte cells compared to corneal epithelial cells. The specific genes were keratocan, lumicane, aldehyde dehydrogenase three members of family A1 (ALDH3A1), and CD34. In addition, immunocytochemistry (ICC) was used to confirm the expression of specific keratocan and lumican markers. Keratocytes was isolated and cultured in the culture medium containing amniotic membrane extract. Based on analyses, keratocan, lumicane, ALDH3A1, and CD34 gene expression in keratocytes was significantly higher than in the epithelial cells. Moreover, keratocan and lumican expression was detected in 92.5% and 91.1% of the cells, respectively. According to the results, the addition of amniotic membrane extract significantly increased the growth of keratocytes. Our findings in this study showed that discarded corneal tissue can be used as a suitable source for obtaining keratocyte cells needed in corneal tissue engineering.

In insect immunity, antibacterial proteins are an important part of the immune system. These proteins are mostly produced by epithelial cells and released through hemolymph. Antibacterial proteins in insects belong to the five major families of cecropins, defensins, attacinlike proteins, proline-rich peptides, and lysozymes. Considering the importance of these proteins in fighting infections, the aim of this study was to evaluate and identify these proteins in silkworms (Bombyx mori). In the present work, profiling of the proteins present in the hemolymph of control silkworms versus, those infected with bacteria was performed by SDS-PAGE, 2D gel electrophoresis, and image analysis. We also used MALDI-TOF and MS/MS to investigate novel and uncharacterized immune protein. For this aim, the silkworm hemolymph after inoculation and infection with Staphylococcus aureus bacteria was analyzed using SDS-PAGE, 2-dimensional gel electrophoresis, MALDI-TOF and MS/MS to identify the immune proteins. The Swiss-Prot and NCBI databases were used for protein identification. A novel protein with a molecular weight of 31.9 KDa was discovered on the fourth day of exposure to bacteria. The expressed protein showed effective activity against S. aureus, which was infected silkworms. In the MALDI-TOF/MS result and protein identification analysis, 90 numbers of mass values were searched, the mass values matched 16, the total sequence coverage was 46%, and their score was 57. According to the analyses, the expressed protein belongs to the hemolysin secretion protein (HlyD). The results led to the identification of a new protein with antimicrobial properties in silkworm, although more information is needed.

Cancer is a complex disease influenced by genetics and environmental factors, registering a high annual mortality rate worldwide. Despite significant advances in cancer treatment, conventional drug therapies for various cancers have many side effects. Today, the use of complementary medicine is prevalent. Curcumin, as a polyphenol, has many biological activities such as antioxidant, anti-inflammatory, antimicrobial, and antiviral activity. In recent years, its anti-cancer potential has been recognized by scientists worldwide. In the present study, the toxicity of diethylhexyl phthalate (DEHP) and the inhibitory effect of curcumin on the expression of matrix metalloproteinases (MMPs) 1, 8, 13, and 18 as genes involved in metastasis in RAW264.7 cell line were investigated. In this study, the effect of different doses of DEHP and curcumin, were respectively, studied on the cancer cell line ofRAW264.7 by MTT assay and AO/EB staining. The RT PCR was employed to determine the gene expression of MMPs 1, 8, 13, and 18. The results of the MTT assay and AO/EB staining indicated that the optimal dose of DEHP was 200 μM, and the optimal dose of curcumin was 25 μM. The combination group selected the same dose (optimal dose of DEHP and curcumin) The results of Real-time PCR showed a decrease in the expression of curcumin-influenced MMPs 1, 8, 13, and 18 genes. The in vitro inhibitory effect of curcumin on the expression of MMPs genes, which are very important in cancer cell metastasis and establishment, encourages us to continue this project in the form of in vivo research.

Troponin enzyme is a gold biomarker for detecting heart attacks. IF it is possible to measure blood troponin levels at the onset of heart pain or symptoms of myocardial infarction, heart damage can be determined by measuring how much it changes. Diagnostic kits are available on the market to detect the presence of enzymes in the blood, but these kits do not check the extent of enzymatic changes. Actually, these kits only check for more than a certain amount of enzyme in the blood. Many experiments can be performed with the advent of lab-onchips. In the proposed method, the troponin enzyme is separated from the rest of the blood by the selected aptamer and then the concentration of the troponin enzyme in the sample is measured by the electrochemical impedance method and Arduino board and coding. The Arduino software was used for coding and simulation, and electrochemical spectroscopy was performed by simulating the behavior of an electrochemical sensor. According to the findings of the present study, the initial measurement by the device showed an error of 55%, which was reduced to 13.5% in the measurement by changing the measuring factors. The manufactured device has the ability to receive the sample, separate it into essential and non-essential components, extract the required information from the sample and also analyze the obtained information. However, the electronic structural factors of the device such as resistance, etc. but must be changed in order to reach 95% reliability.