The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications

Single-stranded DNA binding proteins (SSBs) bind to single-stranded DNA in a sequence-independent manner to prevent formation of secondary structures and protect DNA from nuclease degradation. These ubiquitous proteins are present in prokaryotes, eukaryotes, and viruses, and play a pivotal role in t...

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Main Authors: Olesia Werbowy, Aleksandra Stefańska-Kaźmierczak, Agata Jurczak-Kurek, Karolina Kwiatkowska-Semrau, Anna-Karina Kaczorowska, Magdalena Płotka, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Slawomir Dąbrowski, Tadeusz Kaczorowski
Format: Article
Language:English
Published: MDPI AG 2020-07-01
Series:Proceedings
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Online Access:https://www.mdpi.com/2504-3900/50/1/135
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spelling doaj-07d5fd7c090c464ab0dcdc176f017a2e2020-11-25T03:33:32ZengMDPI AGProceedings2504-39002020-07-015013513510.3390/proceedings2020050135The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech ApplicationsOlesia Werbowy0Aleksandra Stefańska-Kaźmierczak1Agata Jurczak-Kurek2Karolina Kwiatkowska-Semrau3Anna-Karina Kaczorowska4Magdalena Płotka5Olafur H. Fridjonsson6Gudmundur O. Hreggvidsson7Slawomir Dąbrowski8Tadeusz Kaczorowski9Laboratory of Extremophiles Biology, Department of Microbiology, University of Gdansk, 80-308 Gdansk, PolandA&A Biotechnology, 81-451 Gdynia, PolandDepartment of Molecular Evolution, University of Gdansk, 80-308 Gdansk, PolandLaboratory of Extremophiles Biology, Department of Microbiology, University of Gdansk, 80-308 Gdansk, PolandCollection of Plasmids and Microorganisms, University of Gdansk, 80-309 Gdansk, PolandLaboratory of Extremophiles Biology, Department of Microbiology, University of Gdansk, 80-308 Gdansk, PolandMatis, 12 113 Reykjavik, IcelandMatis, 12 113 Reykjavik, IcelandA&A Biotechnology, 81-451 Gdynia, PolandLaboratory of Extremophiles Biology, Department of Microbiology, University of Gdansk, 80-308 Gdansk, PolandSingle-stranded DNA binding proteins (SSBs) bind to single-stranded DNA in a sequence-independent manner to prevent formation of secondary structures and protect DNA from nuclease degradation. These ubiquitous proteins are present in prokaryotes, eukaryotes, and viruses, and play a pivotal role in the following major cellular processes: replication, recombination, and repair of genetic material. In DNA replication, SSB proteins specifically stimulate DNA polymerase, increase fidelity of DNA synthesis, assist the advance of DNA polymerase, and organize and stabilize replication forks. Here, we present our characterization of four SSB proteins of different origins. One of them was isolated from <i>Clostridium</i> sp. phage phiCP130 (SSB C1: 124 aa, Mr = 13,905). Three others (SSB M2: 136 aa, Mr = 15,009; SSB M3: 144 aa, Mr = 16,106; and SSB M5: 138 aa, Mr = 15,851) were isolated from metagenomics libraries. They show high similarity to SSB proteins from <i>Caldanaerovirga</i><i> </i><i>acetigigens</i>, <i>Caldanaerobius</i><i> </i><i>fijiensis</i>, and <i>Fervidobacterium</i><i> </i><i>gondwanense</i>. The recombinant proteins were overproduced in <i>E. coli</i> Rosetta (pRARE), except for SSB M5, which was overproduced in <i>E. coli</i> BL21. Proteins were purified using a metal-affinity chromatography as His-tagged fusion proteins. Electrophoretic mobility shift assay was used to examine their DNA binding activity with fluorescein-labeled oligonucleotide (dT40) used as a substrate. Thermal stability analysis revealed that they are stable at elevated temperatures, with the exception of SSB protein C1, which loses its activity above 65 °C. The other proteins are active at high temperatures, SSB M3 up to 85 °C, while SSB M2 and SSB M5 are active up to 98.7 °C. The subunit structure of proteins was analyzed by gel filtration on Superdex 75 column (AKTA). This allowed us to conclude that in solution, the analyzed proteins exist in oligomeric form, a feature which is characteristic of other SSB proteins. Purified SSB proteins were tested to improve specificity of PCR-based DNA amplification.https://www.mdpi.com/2504-3900/50/1/135DNA-binding proteinsingle-stranded DNAoligomers
collection DOAJ
language English
format Article
sources DOAJ
author Olesia Werbowy
Aleksandra Stefańska-Kaźmierczak
Agata Jurczak-Kurek
Karolina Kwiatkowska-Semrau
Anna-Karina Kaczorowska
Magdalena Płotka
Olafur H. Fridjonsson
Gudmundur O. Hreggvidsson
Slawomir Dąbrowski
Tadeusz Kaczorowski
spellingShingle Olesia Werbowy
Aleksandra Stefańska-Kaźmierczak
Agata Jurczak-Kurek
Karolina Kwiatkowska-Semrau
Anna-Karina Kaczorowska
Magdalena Płotka
Olafur H. Fridjonsson
Gudmundur O. Hreggvidsson
Slawomir Dąbrowski
Tadeusz Kaczorowski
The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications
Proceedings
DNA-binding protein
single-stranded DNA
oligomers
author_facet Olesia Werbowy
Aleksandra Stefańska-Kaźmierczak
Agata Jurczak-Kurek
Karolina Kwiatkowska-Semrau
Anna-Karina Kaczorowska
Magdalena Płotka
Olafur H. Fridjonsson
Gudmundur O. Hreggvidsson
Slawomir Dąbrowski
Tadeusz Kaczorowski
author_sort Olesia Werbowy
title The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications
title_short The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications
title_full The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications
title_fullStr The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications
title_full_unstemmed The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications
title_sort characteristics of new ssb proteins from metagenomic libraries and their use in biotech applications
publisher MDPI AG
series Proceedings
issn 2504-3900
publishDate 2020-07-01
description Single-stranded DNA binding proteins (SSBs) bind to single-stranded DNA in a sequence-independent manner to prevent formation of secondary structures and protect DNA from nuclease degradation. These ubiquitous proteins are present in prokaryotes, eukaryotes, and viruses, and play a pivotal role in the following major cellular processes: replication, recombination, and repair of genetic material. In DNA replication, SSB proteins specifically stimulate DNA polymerase, increase fidelity of DNA synthesis, assist the advance of DNA polymerase, and organize and stabilize replication forks. Here, we present our characterization of four SSB proteins of different origins. One of them was isolated from <i>Clostridium</i> sp. phage phiCP130 (SSB C1: 124 aa, Mr = 13,905). Three others (SSB M2: 136 aa, Mr = 15,009; SSB M3: 144 aa, Mr = 16,106; and SSB M5: 138 aa, Mr = 15,851) were isolated from metagenomics libraries. They show high similarity to SSB proteins from <i>Caldanaerovirga</i><i> </i><i>acetigigens</i>, <i>Caldanaerobius</i><i> </i><i>fijiensis</i>, and <i>Fervidobacterium</i><i> </i><i>gondwanense</i>. The recombinant proteins were overproduced in <i>E. coli</i> Rosetta (pRARE), except for SSB M5, which was overproduced in <i>E. coli</i> BL21. Proteins were purified using a metal-affinity chromatography as His-tagged fusion proteins. Electrophoretic mobility shift assay was used to examine their DNA binding activity with fluorescein-labeled oligonucleotide (dT40) used as a substrate. Thermal stability analysis revealed that they are stable at elevated temperatures, with the exception of SSB protein C1, which loses its activity above 65 °C. The other proteins are active at high temperatures, SSB M3 up to 85 °C, while SSB M2 and SSB M5 are active up to 98.7 °C. The subunit structure of proteins was analyzed by gel filtration on Superdex 75 column (AKTA). This allowed us to conclude that in solution, the analyzed proteins exist in oligomeric form, a feature which is characteristic of other SSB proteins. Purified SSB proteins were tested to improve specificity of PCR-based DNA amplification.
topic DNA-binding protein
single-stranded DNA
oligomers
url https://www.mdpi.com/2504-3900/50/1/135
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