Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts : A promising technology for SUMO fusion proteins
Copyright © 2023 Elsevier B.V. All rights reserved..
The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2024 |
|---|---|
| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:255 |
|---|---|
| Enthalten in: |
International journal of biological macromolecules - 255(2024) vom: 20. Jan., Seite 128258 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Babbal [VerfasserIn] |
|---|
| Links: |
|---|
| Anmerkungen: |
Date Completed 10.01.2024 Date Revised 10.01.2024 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1016/j.ijbiomac.2023.128258 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM364759682 |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM364759682 | ||
| 003 | DE-627 | ||
| 005 | 20240114233422.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231226s2024 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1016/j.ijbiomac.2023.128258 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1255.xml |
| 035 | |a (DE-627)NLM364759682 | ||
| 035 | |a (NLM)37984574 | ||
| 035 | |a (PII)S0141-8130(23)05157-7 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Babbal |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts |b A promising technology for SUMO fusion proteins |
| 264 | 1 | |c 2024 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 10.01.2024 | ||
| 500 | |a Date Revised 10.01.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2023 Elsevier B.V. All rights reserved. | ||
| 520 | |a The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Enzyme immobilization | |
| 650 | 4 | |a Enzyme recycling | |
| 650 | 4 | |a Iron oxide nanoparticles | |
| 650 | 4 | |a SUMO protease | |
| 650 | 4 | |a Whole-cell biocatalyst | |
| 650 | 7 | |a Small Ubiquitin-Related Modifier Proteins |2 NLM | |
| 650 | 7 | |a Peptide Hydrolases |2 NLM | |
| 650 | 7 | |a EC 3.4.- |2 NLM | |
| 650 | 7 | |a Enzymes, Immobilized |2 NLM | |
| 650 | 7 | |a Chitosan |2 NLM | |
| 650 | 7 | |a 9012-76-4 |2 NLM | |
| 650 | 7 | |a Cysteine Endopeptidases |2 NLM | |
| 650 | 7 | |a EC 3.4.22.- |2 NLM | |
| 650 | 7 | |a Recombinant Fusion Proteins |2 NLM | |
| 700 | 1 | |a Mohanty, Shilpa |e verfasserin |4 aut | |
| 700 | 1 | |a Khasa, Yogender Pal |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t International journal of biological macromolecules |d 1992 |g 255(2024) vom: 20. Jan., Seite 128258 |w (DE-627)NLM012627356 |x 1879-0003 |7 nnns |
| 773 | 1 | 8 | |g volume:255 |g year:2024 |g day:20 |g month:01 |g pages:128258 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1016/j.ijbiomac.2023.128258 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 255 |j 2024 |b 20 |c 01 |h 128258 | ||