The histidyl-trna synthetase from streptococcus equisimilis: Overexpression in escherichia coli, purification, and characterization

Corazon A. Menguito, John Papaconstantinou, Paul H. Weigel

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene (hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7–7, thereby creating a fusion gene construct (pT7–7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGPl-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of -18% of total cell protein (-50 mg/liter of bacterial culture). The fusion HisRS was purified to >99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the funtional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of Mr = 53.7 kDa.

Original languageEnglish (US)
Pages (from-to)449-472
Number of pages24
JournalPreparative Biochemistry
Volume23
Issue number4
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Genetics

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