Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/XhoI, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using NcoI/XhoI resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2020 |
|---|---|
| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:69 |
|---|---|
| Enthalten in: |
BioTechniques - 69(2020), 3 vom: 16. Sept., Seite 161-170 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Tham, Hong Y [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
Antimicrobial activity |
|---|
| Anmerkungen: |
Date Completed 09.08.2021 Date Revised 09.08.2021 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.2144/btn-2020-0034 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM313622930 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM313622930 | ||
| 003 | DE-627 | ||
| 005 | 20231225150954.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2020 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.2144/btn-2020-0034 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1045.xml |
| 035 | |a (DE-627)NLM313622930 | ||
| 035 | |a (NLM)32787565 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Tham, Hong Y |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin |
| 264 | 1 | |c 2020 | |
| 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 09.08.2021 | ||
| 500 | |a Date Revised 09.08.2021 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/XhoI, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using NcoI/XhoI resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a MRSA | |
| 650 | 4 | |a antimicrobial activity | |
| 650 | 4 | |a endolysin | |
| 650 | 4 | |a inclusion body | |
| 650 | 4 | |a pET28a | |
| 650 | 4 | |a protein expression | |
| 650 | 7 | |a Recombinant Proteins |2 NLM | |
| 650 | 7 | |a Endopeptidases |2 NLM | |
| 650 | 7 | |a EC 3.4.- |2 NLM | |
| 650 | 7 | |a endolysin |2 NLM | |
| 650 | 7 | |a EC 3.4.99.- |2 NLM | |
| 700 | 1 | |a Song, Adelene A-L |e verfasserin |4 aut | |
| 700 | 1 | |a Yusoff, Khatijah |e verfasserin |4 aut | |
| 700 | 1 | |a Tan, Geok H |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t BioTechniques |d 1988 |g 69(2020), 3 vom: 16. Sept., Seite 161-170 |w (DE-627)NLM012627046 |x 1940-9818 |7 nnns |
| 773 | 1 | 8 | |g volume:69 |g year:2020 |g number:3 |g day:16 |g month:09 |g pages:161-170 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.2144/btn-2020-0034 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 69 |j 2020 |e 3 |b 16 |c 09 |h 161-170 | ||