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Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification.
Protein Expr Purif. 1999 Jun; 16(1):96-102.PE

Abstract

Polymerase chain reaction (PCR)-derived DNA fragment containing the complete structural gene for SSB protein of the Escherichia coli was cloned into an expression vector. The clones expressing His-tagged SSB protein were selected. The cloned DNA fragments were verified to be authentic by sequencing several clones. The recombinant SSB protein (His-tagged SSB) contained a polyhistidine tag at the N-terminus (38 additional amino acids) that allowed single-step isolation by Ni2+ affinity chromatography. We found that recombinant plasmids are unstable and give a low level of expression in E. coli BL21(DE3) strain. However, the plasmids were stable in E. coli BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were highly expressed upon IPTG addition. The SSB protein was purified by metal-affinity chromatography on Ni2+-TED-Sepharose columns. The enzyme was characterized by fluorescence titration experiments for single-stranded DNA binding activity. We have applied the use of His-tagged SSB protein to increase amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving PCR efficiency.

Authors+Show Affiliations

Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, Gdańsk, 80-952, Poland.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10336866

Citation

Dabrowski, S, and J Kur. "Cloning, Overexpression, and Purification of the Recombinant His-tagged SSB Protein of Escherichia Coli and Use in Polymerase Chain Reaction Amplification." Protein Expression and Purification, vol. 16, no. 1, 1999, pp. 96-102.
Dabrowski S, Kur J. Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification. Protein Expr Purif. 1999;16(1):96-102.
Dabrowski, S., & Kur, J. (1999). Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification. Protein Expression and Purification, 16(1), 96-102.
Dabrowski S, Kur J. Cloning, Overexpression, and Purification of the Recombinant His-tagged SSB Protein of Escherichia Coli and Use in Polymerase Chain Reaction Amplification. Protein Expr Purif. 1999;16(1):96-102. PubMed PMID: 10336866.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification. AU - Dabrowski,S, AU - Kur,J, PY - 1999/5/25/pubmed PY - 1999/5/25/medline PY - 1999/5/25/entrez SP - 96 EP - 102 JF - Protein expression and purification JO - Protein Expr Purif VL - 16 IS - 1 N2 - Polymerase chain reaction (PCR)-derived DNA fragment containing the complete structural gene for SSB protein of the Escherichia coli was cloned into an expression vector. The clones expressing His-tagged SSB protein were selected. The cloned DNA fragments were verified to be authentic by sequencing several clones. The recombinant SSB protein (His-tagged SSB) contained a polyhistidine tag at the N-terminus (38 additional amino acids) that allowed single-step isolation by Ni2+ affinity chromatography. We found that recombinant plasmids are unstable and give a low level of expression in E. coli BL21(DE3) strain. However, the plasmids were stable in E. coli BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were highly expressed upon IPTG addition. The SSB protein was purified by metal-affinity chromatography on Ni2+-TED-Sepharose columns. The enzyme was characterized by fluorescence titration experiments for single-stranded DNA binding activity. We have applied the use of His-tagged SSB protein to increase amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving PCR efficiency. SN - 1046-5928 UR - https://www.unboundmedicine.com/medline/citation/10336866/Cloning_overexpression_and_purification_of_the_recombinant_His_tagged_SSB_protein_of_Escherichia_coli_and_use_in_polymerase_chain_reaction_amplification_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(99)91044-6 DB - PRIME DP - Unbound Medicine ER -