A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications

Skowron, Piotr M.; Krawczun, Natalia; Żebrowska, Joanna; Krefft, Daria; Żołnierkiewicz, Olga; Bielawa, Marta; Jeżewska-Frąckowiak, Joanna; Janus, Łukasz; Witkowska, Małgorzata; Palczewska, Małgorzata; Schumacher, Adriana; Wardowska, Anna; Deptuła, Milena; Czupryn, Artur; Mucha, Piotr; Piotrowski, Arkadiusz; Sachadyn, Paweł; Rodziewicz‐Motowidło, Sylwia; Pikuła, Michał; Żylicz-Stachula, Agnieszka

doi:10.1016/j.msec.2019.110426

Cited by 9 publications

(26 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cell death was normalized with respect to untreated control (without IM). Non-IM treated cells were used as negative control (0%) and Triton-X 100 (1%) was used as the positive control for maximum LDH release (maximum cytotoxicity, 100%) [ 26 , 62 ]. The assay was performed to IM in concentrations of 50–150 µg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…The medium was then changed, again, and cells were stimulated with an appropriate concentration of IM. After 24 h the cells were fixed with 3.7% paraformaldehyde, stained with 0.05% crystal violet, and the effect was measured with a phase-contrast fluorescent microscope (Zeiss, Oberkochen, Germany) and the surface areas were counted with Imaging Software NIS-Element Basic Research program [ 26 , 62 ].…”

Section: Methodsmentioning

confidence: 99%

“…10 4 cells/100 µl were stained with monoclonal, fluorochrome-conjugated antibodies (anti-CD3, -CD4, -CD8, -CD16, -CD56, -CD25, -CD69, -CD71, -HLA-DR, -CD11c, -CD80, and -CD83) and incubated for 30 min in RT in the dark. Stained cells were analyzed using a flow cytometer (LSRFortessa, BD, USA) [ 26 , 62 ]. All data are presented as percentages due to the application of hospital diagnostic laboratory standards for immune cells analyses.…”

Section: Methodsmentioning

confidence: 99%

“…Then, erythrocyte lysis was performed, the cells were washed and analyzed by flow cytometry (BD FACSCanto II, San Jose, CA, USA). Each sample was accompanied by its own negative and positive control (anti-FcεRI mAb and fMLP) [ 62 , 65 ].…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Imunofan—RDKVYR Peptide—Stimulates Skin Cell Proliferation and Promotes Tissue Repair

et al. 2020

Self Cite

View full text Add to dashboard Cite

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today’s science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name “Imunofan” (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30–40% and 20–50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Imunofan—RDKVYR Peptide—Stimulates Skin Cell Proliferation and Promotes Tissue Repair

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…To address this gap, here, we have explored a new strategy for the production of a recombinant antimicrobial protein based on a multidomain polypeptide that combines different functional domains in a single molecule but without a carrier protein. The combination of several domains has been previously reported for other proteins [20][21][22][23], but none of them for antimicrobial treatment purposes. Additionally, taking into consideration the specific requirements of price, stability, toxicity, effectiveness, and delivery that appear to be key parameters in the development of a new generation of antimicrobials [3], we have added aggregation-seeding domains based on leucine zippers that increase the recombinant production of the antimicrobial molecules as protein nanoclusters (also known as inclusion bodies (IBs)) [24].…”

Section: Introductionmentioning

confidence: 99%

A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity

et al. 2020

View full text Add to dashboard Cite

Background: Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity. Results: A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA 2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM. Conclusions: Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.

show abstract

Data regarding a new, vector-enzymatic DNA fragment amplification-expression technology for the construction of artificial, concatemeric DNA, RNA and proteins, as well as biological effects of selected polypeptides obtained using this method

et al. 2020

View full text Add to dashboard Cite

A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications

Cited by 9 publications

References 30 publications

Imunofan—RDKVYR Peptide—Stimulates Skin Cell Proliferation and Promotes Tissue Repair

Imunofan—RDKVYR Peptide—Stimulates Skin Cell Proliferation and Promotes Tissue Repair

A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity

Data regarding a new, vector-enzymatic DNA fragment amplification-expression technology for the construction of artificial, concatemeric DNA, RNA and proteins, as well as biological effects of selected polypeptides obtained using this method

Contact Info

Product

Resources

About