Expression of the Mouse HSP27 Chaperone in CHO-K1 Cells for the Enhancement of Viable Cell Density in Batch Culture

Mohammad Reza Amini, Azam Rahimpour, Reyhaneh Hoseinpoor, Masoumeh Rajabibazl

Trends in Peptide and Protein Sciences, Vol. 7 (2022), , Page 1-5 (e2)
https://doi.org/10.22037/tpps.v7i.37418

Chinese hamster ovary (CHO) cells are extremely vulnerable to cell viability loss in culture despite the availability of different nutrients supplementation strategies. As a result, extending the culture lifetime can profoundly increase recombinant protein expression. Overexpression of HSP27 and its anti-apoptotic effects have been shown in human cell lines in previous studies. In the current study, mouse HSP27 (mHSP27) was cloned in pcDNA 3.1 hygro expression vector and was expressed in CHO-K1 cells to assess its impacts on cell viability and growth. Expression of mHSP27 in CHO-K1 cells was confirmed using RT-PCR. A 3-fold enhancement in peak viable cell density of mHSP27 transfected clones was observed, and culture viability loss was delayed by 2 days compared to un-transfected cells. In future studies, the resulting mHSP27 CHO-K1 cells could be employed as a novel host system for the transient and stable expression of therapeutic recombinant proteins.


HIGHLIGHTS


  • Cell engineering is an effective strategy to cope with apoptosis in CHO cells.

  • HSP27 is involved in mammalian cell apoptosis.

  • Expression of the mouse HSP27 increased the viability and cell density of CHO-K1 cells.

Preparation and In Vitro Characterization of Crocin-loaded Casein Hydrogels

Fatemeh Mehryab, Fatemeh Taghizadeh, Azadeh Haeri

Trends in Peptide and Protein Sciences, Vol. 7 (2022), , Page 1-9 (e3)
https://doi.org/10.22037/tpps.v7i.37420

Crocin, the main active constituent of saffron, has many important biological activities. Due to its anti-inflammatory properties, crocin can be potentially effective in different pathological conditions including oral ulcers. Novel drug delivery systems such as hydrogels have been used to increase the stability of crocin and provide a controlled release of this compound. Casein is the main protein of milk that possesses suitable properties for the fabrication of hydrogels. In this paper, casein-based hydrogels with different casein to crocin weight ratios were synthesized using the acid-gelation method. The prepared crocin-loaded hydrogels were characterized regarding their rheological behavior, drug content, swelling ratio, surface morphology, thermal stability, and in vitro release profile. The structure of casein hydrogels was characterized using Fourier transform infrared and X-ray diffraction. All formulations exhibited a pseudoplastic rheological behavior and there was no statistically significant difference in viscosity among them. Hydrogel with casein to crocin weight ratio of 10:1 had larger pores and demonstrated a higher swelling percentage and suitable thermal stability. All casein-based hydrogels demonstrated a slow release of crocin over 24 hours and the hydrogel with lower casein to crocin weight ratio had an increased release rate. Taken together, casein-based hydrogels were found to be effective carriers to provide a controlled release system for crocin delivery.


HIGHLIGHTS


  • Casein-based hydrogels were developed for delivery of crocin.

  • Casein-based hydrogels provided a controlled in vitro release profile for crocin.

  • Hydrogel with a lower casein ratio exhibited a higher release rate of crocin.

Controversy Between In Vitro Biological Activities of a Novel Designed Antimicrobial Peptide and Its In Silico Predicted Activities

Fariba Fathi , Maryam Ghobeh , Arash Mahboubi, Maryam Tabarzad

Trends in Peptide and Protein Sciences, Vol. 7 (2022), , Page 1-12 (e4)
https://doi.org/10.22037/tpps.v7i.38346

Due to their unique mechanisms of action, antimicrobial peptides (AMPs) are promising candidates to combat different infectious diseases. They usually non-specifically interact with the bacterial cell membrane, create pores in their membrane and increase its permeability which causes the death of pathogens. In the design and development of AMPs, in silico strategies have been developed to enhance the function and activity of natural peptides. In this study, in silico approaches were used to develop a novel AMP with several extra bioactivities. Then, the designed AMP were analyzed through computational methods by in vitro experiments. Bioinformatics research revealed a 10-amino-acid peptide (LVSARIRCPK) having antibacterial, anti-biofilm, antiviral, antifungal, and anti-inflammatory effects. However, only the antiviral capabilities of the peptide were validated in the experimental analysis of antibacterial, antifungal, and antiviral activities. This data suggests that; while bioinformatics approaches have greatly advanced in recent years, more optimization work has to be done in order to attain high accuracy and minimize mistakes.


HIGHLIGHTS


  • A novel 10 residues anti-microbial peptide was designed using bioinformatics tools.

  • In vitro analysis showed that this novel AMP did not have efficient antimicrobial activities.

  • Stringencies of bioinformatics criteria thresholds setting may result in better design.

Since the appearance of acquired immunodeficiency syndrome (AIDS) disease, millions of people got infected, thousands are died and many suffered from its complications. One of the chronic and late symptoms of AIDS is lipodystrophy that leads to losing of fat in some parts of the body while gaining it at other organs and sites. One of the main targets in drug development for management of lipodystrophy is growth hormone-releasing hormone (GHRH) receptor. As a secondary metabolite from plants, leginsulin is a peptide with 37 amino acids and considered as hormone-like peptide. In this study, through in silico approaches, binding of leginsulin and GHRH receptor was studied. The results showed strong binding of the two molecules with docking score of -324.16 and ligand RMSD of 47.26. The molecular dynamic investigation also revealed these two proteins remained bound for almost 104 ns. Evaluation of the peptide toxicity in the body had shown that it is not toxic to the human organs and also, it doesn’t pass through the blood brain barrier. The results support the use of legumes as a source of leginsulin for potential management of lipodystrophy in the patients with AIDS.


HIGHLIGHTS


  • Leginsulin is a plant secondary metabolite with 37 amino acids peptide structure.

  • Binding of leginsulin and GHRH receptor was studied through in silico approaches.

  • Results support the leginsulin as a potential management of lipodystrophy in AIDS patients.

Antioxidant Activity of Peptides Derived from Enzymatic Digestion of Spirulina platensis Protein Extract by Different Proteases

Hanieh Niknam, Fariba Fathi, Arash Mahboubi, Maryam Tabarzad

Trends in Peptide and Protein Sciences, Vol. 7 (2022), , Page 1-7 (e6)
https://doi.org/10.22037/tpps.v7i.38965

One of the attractive sources of bioactive compounds is cyanobacteria and in particular, Chlorella vulgaris and Spirulina platensis. Enzymatic digestion of the Spirulina protein extract can result in bioactive peptides with diverse activities, including antioxidant function. This study aims to produce peptides with antioxidant properties after the enzymatic digestion of Spirulina platensis protein extract using three enzymes: bacterial protease, pepsin, and papain. The protein extract from Spirulina platensis was subjected to enzyme hydrolysis for 3 hours at 37°C (pH 7.4 for papain and bacterial protease and pH 5 for pepsin). The concentration of peptide fragments was evaluated to determine the yield of protein digestion. In order to measure the level of anti-oxidative potential of the hydrolysates, the DPPH assay was run. The results indicated that the bacterial protease led to the highest concentration of peptide fragments, while the hydrolysate obtained from pepsin digestion showed the most antioxidant activity (80%), mainly the peptides that have molecular weights less than 14 KDa. Consequently, pepsin can be a proper enzyme to produce antioxidant peptides from the protein extract of S. platensis. In conclusion, the results of the study confirmed that the products of enzymatic digestion by different enzymes have distinct features.


HIGHLIGHTS


  • Spirulina platensis protein extract was digested with three types of protease.

  • Digestion of protein extract by pepsin resulted in higher antioxidant activity.

  • Digestion by bacterial protease from Bacillus licheniformis resulted in higher yield of peptide formation.

Cytoplasmic Expression of Human Bone Morphogenetic Protein-7 by a Genetically Engineered Strain of Escherichia coli, SHuffle® Strain

Alireza Dugmehchi, Ghazal Sadipour, Yeganeh Talebkhan, Hoda Jahandar, Fahimeh Nemati, Elham Mohit, Leila Nematollahi

Trends in Peptide and Protein Sciences, Vol. 7 (2022), , Page 1-7 (e7)
https://doi.org/10.22037/tpps.v7i.39039

Homodimeric bone morphogenetic protein-7 (BMP-7) plays a key role in bone metabolism. The functionality of human BMP-7 protein is dependent on its disulfide bond formation and proper folding. Therefore, the expression of soluble recombinant BMP-7 using Escherichia coli cells as the host remains a challenge. Given the need for these disulfide-bonded proteins for stabilized native conformation, the cytoplasm of SHuffle® T7 Express as an E. coli engineered strain can effectively fold disulfide-bonded proteins with a need for proper oxidative folding. These cellular features turn the SHuffle® expression system into an efficient host for the recombinant production of human BMP-7 protein. A soluble dimeric form of recombinant human BMP-7 (rhBMP-7) which has a wide range of applications in medicine and can be used in the treatment of bone defects was produced using the SHuffle® strain as the expression system. This study demonstrated the production of rhBMP-7 using E. coli SHuffle® T7 Express strain. Also, an effective protocol was proposed for the expression and purification of soluble human BMP-7. In addition, it was found that the genetically engineered SHuffle® strain can efficiently enhance the solubility of recombinant human BMP-7 as a therapeutic target.


HIGHLIGHTS                                                             


  • E. coli SHuffle® T7 Express strain is an effective host to express disulfide-bonded proteins.

  • BMP-7 is involved in the process of bone formation.

  • Expression of human BMP-7 in SHuffle® strain increased its solubility.

 

Synthesis, Radiolabeling and Stability Studies of Peptide HYNIC-LIKKP-Pyr-F with 99mTc as an Apoptosis Imaging Agent

Elmira Javani Jouni, Mohammadsaeed Kordi, Sepideh Khoshbakht, Salimeh Amidi , Soraya Shahhosseini

Trends in Peptide and Protein Sciences, Vol. 7 (2022), , Page 1-5 (e8)
https://doi.org/10.22037/tpps.v7i.39024

A non-invasive method for detecting phosphatidylserine (PS) exposure on the outer surface of plasma membranes, such as nuclear imaging, could aid in the diagnosis and treatment of diseases associated with apoptosis. Annexin V has been the most researched imaging agent for apoptosis to date. Due to Annexin V's limitations, additional agents, such as small peptides and molecules, have been introduced, including LIKKPF developed by Burtea et al. In this study, HYNIC-LIKKP-pyr-F, a derivative of LIKKPF was prepared using the 9-fluoroenylmethoxycarbonyl (fmoc) method, radiolabeled with Technetium-99m (99mTc) with the use of Stannous chloride (SnCl2) as a reducing agent and ethylenediamine diacetate (EDDA) and tricine as co-ligands. Radiochemical purity, labeling efficiency, and stability of radiopeptide in normal saline and human plasma were determined using thin layer chromatography (TLC). The partition coefficient of radiolabeled peptide was measured in a combination of PBS (pH 7.4) and n-octanol. Specific activity was also measured. LC-MS was used to examine the synthesized peptide. Peptide was stable in human serum for at least 4 hr. Peptide was radiolabeled with 99mTc with radiochemical purity and labeling efficiency over 95% and 90%, respectively. Radiopeptide was stable in saline and human serum for at least 4 hours. The radiolabeled peptide has a great deal of potential as an apoptosis imaging agent for in vitro and in vivo experiments.


HIGHLIGHTS


  • A noninvasive method for apoptosis imaging is the usage of radiolabeled affinity ligands.

  • A new derivative of LIKKPF with affinity for phosphatidyl serine was synthesized.

  • The LIKKPF peptide was radiolabeled with 99m

Carbapenem-resistant and carbapenemase-producing Pseudomonas aeruginosa (CRPA) and methicillin-resistant Staphylococcus aureus (MRSA) are two pathogens that are resistant to currently available antimicrobials. As an alternative to effective medication molecules, antimicrobial peptides (AMPs) have the potential to cure superbug-caused infections effectively. Two new AMPs (ama1 and ama2) were designed utilizing a knowledge-based technique with optimal parameters. First, the PEP-FOLD 3.5 server made a de novo prediction of the AMPs' three-dimensional (3D) structure, which was validated using PROCHECK of SAVES v6.0 by checking the amino acid locations in the Ramachandra plot. Then, protein-peptide docking simulations of the predicted AMPs and reference AMP (Aurein 1.2) for positive control were performed using the HPEPDOCK docking web server, followed by the computation of the AMPs' physicochemical parameters and toxicity profile using the ProtParam and vNN-ADMET web servers, respectively. The sequences for ama1 and ama2 were AWGKIKALR and IKWLRLAKP, respectively. Docking analysis revealed that the antibacterial activity of ama1 and ama2 was superior to that of Aurein 1.2 against CRPA-resistant enzyme (6ew3), respectively. However, ama1, ama2, and Aurein 1.2 inhibited the activity of MRSA-resistant protein (4c12). Both the physicochemical qualities and the toxicity profiles were advantageous. Therefore, the in-silico-derived AMPs could serve as a pharmaceutical candidate for developing multidrug-resistant bacteria-effective antimicrobials.


HIGHLIGHTS


  • Two cationic antimicrobial peptides (AMPs) were designed.

  • Molecular docking of the AMPs revealed better antimicrobial activity than the reference.

  • The novel AMPs had net positive charge and optimal hydrophobic amino acids.

In-house Optimization Radiolabeling of Recombinant scFv with 99mTc-Tricarbonyl and Stability Studies

Safura Joukar, Negar Bozorgchami, Dara Hatamabadi, Abdolreza Yazdani, Elham Mohit, Elmira Javani, Mohammadsaeed Kordi, Sepideh Khoshbakht, Soraya Shahhosseini

Trends in Peptide and Protein Sciences, Vol. 7 (2022), , Page 1-6 (e10)
https://doi.org/10.22037/tpps.v7i.39823

His-tagged scFv fragments of monoclonal antibodies have better pharmacokinetic properties than whole antibodies. Radiolabeled scFvs are considered for targeted imaging and treatment. Technetium tricarbonyl provides radiolabeling of scFvs without losing its biological activity in a fast and easy procedure. Technetium tricabonyl was prepared as follows: A freshly eluted solution of Na99mTcO4 was added to a mixture containing sodium carbonate, sodium potassium tartarate, boranocarbonate, sodium borohydride. The mixture was heated for 30 min at 100°C. Radiochemical purity was determined using radio thin lyer chromatography. Then, technetium tricarbonyl was added to a solution of scFv in PBS buffer and incubated for 2 h at 50°C, purified by PD-10 column and radiochemical purity was determined. Results showed that radiochemical purity of technetium tricarbony was over 98%. The best conditions for radiolabeling of scFv was: scFv concentration >2 mg/mL, PBS buffer, 2 h incubation at 50°C, pH 8-9, and high activity concentration of tricarbonyl. The best radiochemical purity of scFv was 70% before purificarion. Radiolabeled scFv was stable in PBS for 24 h incubation and there was no release of technetium in competition with histidine. In this study, we optimized radiolabeling of a scFv with technetium tricarbonyl using house made boranocarbonates. The results are promising and will be used for future studies.


HIGHLIGHTS


  • Radiolabeling of scFv was done directly by 99mTc-tricarbonyl.

  • 99mTc-tricarbonyl was prepared in house from boranocarbonate.

  • 99mTc-Radiolabeled scFv can be used for radioimmunoscintigraphy.