Chromatographic purification of recombinant human erythropoietin

Adamíková, Jana; Antošová, Monika; Polakovič, Milan

doi:10.1007/s10529-019-02656-8

Cited by 12 publications

(4 citation statements)

References 46 publications

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“…A 10 μl cell lysate, column flow-through, and elution fractions were analyzed on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the purity of protein bands (Tab. S1 ) was measured by densitometry using ImageJ software (v.2.0) (Adamíková et al 2019 ).…”

Section: Methodsmentioning

confidence: 99%

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides

Narh,

Casillas-Vega,

Zarate

2024

Appl Microbiol Biotechnol

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An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) Escherichia coli strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against Staphylococcus aureus, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae bacteria clinical isolates at a lower minimum inhibition concentration level (10–33 μM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50–100 μM). Key points • The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker. • The peptide was expressed with the carrier proteins SmbP and CusF3H+. • The hybrid peptide shows antibacterial potency against clinical bacterial isolates.

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Section: Methodsmentioning

confidence: 99%

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides

Narh,

Casillas-Vega,

Zarate

2024

Appl Microbiol Biotechnol

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“…The conventional methods used for the isolation of cancer-related protein markers are mostly based on different chromatographic approaches, including ion-exchange, size-exclusion, hydrophobic, reverse-phase, and affinity chromatography [218][219][220][221]. Since proteins do not have a generalized purification method, the type of chromatography to be used depends entirely on the physical and chemical properties of the target proteins.…”

Section: Cancer-associated Proteinsmentioning

confidence: 99%

Emerging on‐chip electrokinetic based technologies for purification of circulating cancer biomarkers towards liquid biopsy: A review

Shi

Esfandiari

2021

Electrophoresis

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Early detection of cancer can significantly reduce mortality and save lives. However, the current cancer diagnosis is highly dependent on costly, complex, and invasive procedures. Thus, a great deal of effort has been devoted to exploring new technologies based on liquid biopsy. Since liquid biopsy relies on detection of circulating biomarkers from biofluids, it is critical to isolate highly purified cancer‐related biomarkers, including circulating tumor cells (CTCs), cell‐free nucleic acids (cell‐free DNA and cell‐free RNA), small extracellular vesicles (exosomes), and proteins. The current clinical purification techniques are facing a number of drawbacks including low purity, long processing time, high cost, and difficulties in standardization. Here, we review a promising solution, on‐chip electrokinetic‐based methods, that have the advantage of small sample volume requirement, minimal damage to the biomarkers, rapid, and label‐free criteria. We have also discussed the existing challenges of current on‐chip electrokinetic technologies and suggested potential solutions that may be worthy of future studies.

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“…During purifi cation, all foreign proteins, viruses and toxins have to be removed because they can cause an undesirable immune response. Different chromatographic methods are used for rhEPO purifi cation (Adamíková et al, 2019). Size-exclusion, ion-exchange, hydrophobic, affi nity and multimodal interactions between protein molecules and adsorbent are the most commonly applied in rhEPO purifi cation.…”

Section: Introductionmentioning

confidence: 99%

Recombinant human erythropoietin separation using a cation-exchange multimodal adsorbent

Ostrihoňová

Adamíková

Molnár

et al. 2019

Acta Chimica Slovaca

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This work deals with the capture of human recombinant erythropoietin (rhEPO) from a mixture of proteins in a concentrated postcultivation supernatant. Cation-exchange multimodal adsorbent Capto MMC ImpRes was selected as potential chromatographic separation material. Its equilibrium properties were investigated in batch adsorption experiments. The effect of pH in the range of 5.5—7.5 and NaCl concentration in the range of 0—300 mM on the adsorption of rhEPO and contaminant proteins was examined. Optimal conditions found in these equilibrium experiments were applied to rhEPO adsorption in a chromatographic column. Several experiments were carried out at different elution conditions to optimize the rhEPO yield and selectivity.

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Chromatographic purification of recombinant human erythropoietin

Cited by 12 publications

References 46 publications

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides

Emerging on‐chip electrokinetic based technologies for purification of circulating cancer biomarkers towards liquid biopsy: A review

Recombinant human erythropoietin separation using a cation-exchange multimodal adsorbent

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