Protein and Peptide Letters - Volume 29, Issue 6, 2022

Background: The analysis of biofluid samples with low protein content (e.g., urine or saliva) can be challenging for downstream analysis methods with limited sensitivity. To circumvent this problem, sample processing methods are employed to increase the protein concentration in analyzed samples. However, for some techniques, like differential scanning calorimetry (DSC) that characterizes thermally-induced unfolding of biomolecules, sample processing must not affect native protein structure and stability. Methods: We evaluated centrifugal concentration and stirred cell ultrafiltration, two common methods of sample concentration characterized by a low risk of protein denaturation, with the goal of establishing a protocol for DSC analysis of low concentration biospecimens. Results: Our studies indicate that both methods can affect protein stability assessed by DSC and, even after optimization of several parameters, the obtained DSC profile (thermogram) suggested that sample processing affects the structure or intermolecular interactions of component proteins contributing to altered thermal stability detectable by DSC. We also found a relationship between changes in thermograms and low protein concentration, indicating that diluting biospecimens to concentrations below 0.1 mg/mL can perturb the intermolecular environment and affect the structure of proteins present in the solution. Conclusion: Dilution of samples below 0.1 mg/mL, as well as concentration of samples with low protein content, resulted in affected thermogram shapes suggesting changes in protein stability. This should be taken into account when concentrating dilute samples or employing techniques that lower the protein concentration (e.g., fractionation), when downstream applications include techniques, such as DSC, that require the preservation of native protein forms.

PARP-1 is one of the 18 PARP enzymes that are involved in important processes at the cellular level. The most important tasks of PARP-1 are to detect and repair DNA damage and to prevent processes of apoptosis. By finding and using new strategies for marking and detecting the activity of this protein, it is possible to identify more and more tasks in which it participates. In pathological states, PARP-1 activity increases significantly. Since the 1980s, scientists have been searching for and discussing substances that may inhibit PARP-1 activity and disrupt DNA damage response pathways. In this way, unwanted cells could be destroyed. The paper presents a short description of the methods used in the determination of PARP-1 by various research groups. A critical approach to each of them was also made by pointing to the advantages and disadvantages of the described analytical methods. The literature review contains information on methods useful for PARP-1 determination, such as SPR, QCM, CL and FL, DPV, SDS-PAGE with MS, MALDI MS, Western Blot, ELISA and ATR-FTIR spectroscopy. It also includes analysis of the results of research on inhibitors that may be effective in the diagnosis and treatment of cancer and other diseases.

Background: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5’-phosphorylated end to a 3’-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. Objective: The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases. Methods: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions. Results: In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates. Conclusion: Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.

Background: Interleukin-11 is a pleiotropic cytokine that is known to play an important role in the progression of various forms of cancer by modulating the survival and proliferation of tumour cells. IL11 also demonstrates a structural homology to IL6, the predominant cytokine involved in COVID-19. This makes IL11 a potential therapeutic target in both diseases. Objective: This study aimed to evaluate the impact of the two-point mutations, R135E and R190E, on the stability of IL11 and their effect on the binding affinity of IL11 with its receptor IL11Rα. It is a molecular level analysis based on the existing experimental validation. Methods: Computer-aided drug designing techniques, such as molecular modelling, molecular docking, and molecular dynamics simulations, were employed to determine the consequential effects of the two-point mutations. Results: Our analysis revealed that the two mutations led to a decrease in the overall stability of IL11. This was evident by the increased atomic fluctuations in the mutated regions as well as the corresponding elevation in the deviations seen through RMSD and Rg values. It was also accompanied by a loss in the secondary structural organisation in the mutated proteins. Moreover, mutation R135E led to an increase in the binding affinity of IL11 with IL11Rα and the formation of a more stable complex in comparison to the wild-type protein with its receptor. Conclusion: Mutation R190E led to the formation of a less stable complex than the wild-type, which suggests a decrease in the binding affinity between IL11 and IL11Rα.

Background: Apelin is a newly discovered peptide hormone and originally discovered endogenous apelin receptor ligand. Objective: In this study, we aimed to investigate the possible roles of potassium channel subtypes in the vasorelaxant effect mechanisms of apelin. Methods: The vascular rings obtained from the thoracic aortas of the male Wistar Albino rats were placed into the isolated tissue bath system. The resting tension was set to 2 g. After the equilibration period, the aortic rings were precontracted with 10-5 M phenylephrine (PHE) or 45 mM KCl. Pyroglutamyl-apelin-13 ([Pyr1]apelin-13), which is the dominant apelin isoform in the human cardiovascular tissues and human plasma, was applied cumulatively (10^-10-10-⁶ M) to the aortic rings in the plateau phase. The experimental protocol was repeated in the presence of specific K⁺ channel subtype blockers to determine the role of K⁺ channels in the vasorelaxant effect mechanisms of apelin. Results: [Pyr1]apelin-13 induced a concentration-dependent vasorelaxation (p < 0.001). The maximum relaxation level was approximately 52%, according to PHE-induced contraction. Tetraethylammonium, iberiotoxin, 4-Aminopyridine, glyburide, anandamide, and BaCl2 statistically significantly decreased the vasorelaxant effect level of [Pyr1]apelin-13 (p < 0.001). However, apamin didn’t statistically significantly change the vasorelaxant effect level of [Pyr1]apelin-13. Conclusion: In conclusion, our findings suggest that BKCa, IKCa, Kv, KATP, Kir, and K2P channels are involved in the vasorelaxant effect mechanisms of apelin in the rat thoracic aorta.

Background: Bone morphogenetic protein (BMP) is a cysteine-rich growth factor and plays a key role in early bone tissue development and bone defect repair. However, the low yield, high cost and complicated process in BMP significantly limit its clinical application. Objective: In this study, we developed an efficient method for soluble expression and preparation of recombinant human bone morphogenetic 7-2 fusion protein (rhBMP7-2) and determined its molecular weight and biological activity. Methods: The fusion gene for rhBMP-2 and rhBMP-7 was inserted into the pET-ELP expression vector. Correct DNA sequence was confirmed, the rhBMP7-2-ELP was transformed into Escherichia coli strain BL21 (DE3), and the rhBMP7-2 was produced in the recombinant E. coli. Recombinant BMP7-2 purify was identified using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The cell proliferation and biological activity of rhBMP7-2 were measured by Cell Counting Kit-8 and Alkaline Phosphatase assay using C2C12 cells, respectively. Results: The result of digestion of NdeI, BamHI and XhoI enzymes showed that the rhBMP7-2-ELP was correctly constructed. The recombinant BMP7-2 was successfully expressed in soluble form; the purified rhBMP7-2 showed biological activity and significantly promoted cell proliferation and differentiation in a dose-dependent manner. Conclusion: The rhBMP7-2 fusion protein with osteogenic activity was prepared through a lowcost and time-efficient method. Our preparation method presents the potential to be applied to the large-scale production of rhBMP7-2 and is expected to play a significant role in clinical treatment.

Background: Garlic (Allium sativum) from the family Amaryllidaceae is widely used in culinary and is reported to have potential anticancer, anti-diabetic, antimicrobial, and cardioprotective activities. Allium sativum agglutinin (ASA) is a bulb-type lectin (BTL) domaincontaining lectin isolated from garlic and has been studied for its various biological functions. Previous studies have reported the anti-cancer effects of ASA on histiocytic lymphoma (U937), promyelocytic leukemia (HL60), and oral cancer (KB). Methods: In this study, we have purified and characterized ASA and evaluated it for its anticancer effects on other cancer cell lines. MTT assay and FACS analysis was done to corroborate the anticancer findings against cervical (HeLa) and lung cancer (A549) cell lines. Results: IC50 value of 37 μg/ml in HeLa and a weak activity (26.4 ± 1.9% cellular inhibition at 100μg/ml treatment) in A549 were found in the MTT assay. FACS analysis further corroborated these findings and showed the apoptotic effects of ASA in these cell lines. Conclusion: Anticancer activity for members of bulb-type lectin (BTL) domain-containing lectins has been widely reported, and we hope that our study forms a basis for the development of ASA as a therapeutic agent.