Volume 9, Issue 3 (Summer 2018)
Abstract
Aims: Studies based on thermal stability are considered as one of the methods for investigating the physicochemical properties of proteins in biotechnology. The aim of this study was to evaluate the effect of replacement of Arginine 39 amino acid with lysine on the heat denaturation of mnemiopsin photoprotein 1.
Materials and Methods: In the current experimental study, R39K mutated mnemiopsin was compared with wild protein (in which arginine 39 amino acid was converted to the lysine amino acid). In order to investigate the effect of mutation on the content of the secondary structure, a rotation interpolation method was used. To investigate the possible changes in the rate of thermal stability of mutated and wild proteins, heat denaturation measurements were performed by differential scanning calorimeter. Bioinformatics software were used to compare the structure of two types of proteins.
Findings: The mutated R39K compression decreased in comparison with wild protein. No significant change was observed in the values of thermodynamic parameters, especially Tm. The upward movement of arginine 187 amino acid in the mutated protein decreased the thermal stability of this protein. Increasing the accessible surface of lysine 188 in the mutated protein increased its stability.
Conclusion: In thermal stability of the R39K mutated protein, various factors are effective, including the molecular movements of amino acids, their accessible surface, and the content of the secondary structure of protein stabilizing. This mutation reduces the mutated R39K compression rather than the wild protein; increasing ASA related to Lys188 amino acid in the mutated R39K compared with wild protein increases protein stability, but reducing the amount of secondary structure in this mutated, accompanied by an increase in the molecular upward movement in the Arg187 amino acid serves to reduce the stability of this mutated.
Volume 10, Issue 3 (Summer 2019)
Abstract
CEL I endonuclease pertaining to the S1 endonuclease family. The enzyme, with its high specificity, has the ability to identify different types of mutations and base replacement in the DNA molecule, which makes it important in commercial products to use in research and clinical laboratories. Although the enzyme exists in the celery plant, the extraction of the enzyme is a time-consuming process and not economical and the yield of the final product is low. In addition, due to its post-translational modifications to achieve the final active structure, no report has published to indicate the expression of the active form of this enzyme in the bacterial hosts yet. Therefore, one of the production sources of the active form of this enzyme is its cloning and expression in eukaryotic hosts, including yeast and mammalian cell lines. In this study, in order to express CEL I endonuclease, its gene sequence was optimized and synthesized in host eukaryotic HEK293T. CEL I was subcloned by double digest with KpnI and XhoI enzymes in the pBudCE4.1expression vector. The expression construct was transfected into the HEK293T cell line by lipofectamine transfection. Expression of the recombinant protein after transfection into HEK293T cells was confirmed by multiple methods including polyacrylamide gel electrophoresis, ELISA, RT-PCR, and western blot reaction. The analysis of SDS-PAGE and western blot data confirmed the molecular weight of approximately 30kDa. Purification was carried out with the Ni-NTA column and the amount of purified protein was determined to be about 0.2mg/ml. Finally, the activity of endonuclease enzyme was investigated on both normal and mutated heteroduplex DNA amplified by PCR. The results showed that the expression of this protein in HEK293T host had shown sufficient activity.
Leila Ramezanzade, Seyed Fakhreddin Hosseini, Behrouz Akbari-Adergani, Reza Hasan Sajedi, Anan Yaghmur,
Volume 11, Issue 2 (5-2022)
Abstract
In this study, the orangefin ponyfish (Leiognathus bindus) was hydrolyzed by alcalase in an enzyme to substrate ratio of 1: 100 for 300 minutes, and the degree of hydrolysis was measured for 5 hours. Also, the hydrolysate was fractionated by centrifugal having molecular mass cutoffs of 3, 10, and 30 kDa, and four peptide fractions were obtained. Then, the antioxidant activity (DPPH and ABTS free radicals scavenging activity) of peptide fractions, as well as hydrolysate, were measured at different hydrolysis times. The degree of hydrolysis was the highest (55.43 ± 2.11%) at a hydrolysis time of 240 minutes. The hydrolysate had a high amount of hydrophobic amino acids (50.6%) which cause antioxidant properties. The results of DPPH radical scavenging activity showed that the highest scavenging activity was obtained at a hydrolysis time of 240 minutes (75.59 ± 1.46). Also, among all the fractions, the 3-10 kDa fraction exhibited the highest scavenging activity compared to other fractions (80.58 ± 2.96% at a concentration of 5mg /ml). Based on the result of ABTS radical scavenging, the highest activity was reported at 240 minutes after hydrolysis (50.54 ± 0.63). Also, among all peptide fractions, the 3-10 kDa fraction had significantly higher scavenging activity than other fractions (84.58 ± 0.44 at a concentration of 5 mg /ml). The results of this study showed that the peptides obtained by enzymatic hydrolysis of orangefin ponyfish are a good candidate for providing antioxidant properties.
