Production of active recombinant eIF5A: Reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Jong Hwan Park, Camila A O Dias, Seung Bum Lee, Sandro R. Valentini, Masaaki Sokabe, Christopher S. Fraser, Myung Hee Park

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N ε-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine-or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

Original languageEnglish (US)
Pages (from-to)301-309
Number of pages9
JournalProtein Engineering, Design and Selection
Volume24
Issue number3
DOIs
StatePublished - Mar 2011

Fingerprint

Escherichia coli
Enzymes
Proteins
Recombinant proteins
Cell proliferation
Binding sites
deoxyhypusine hydroxylase
Tissue
Engineers
Ribosomes
Lysine
hypusine
eukaryotic translation initiation factor 5A
Puromycin
Polyamines
Archaea
Eukaryotic Cells
Recombinant Proteins
Eukaryota
Binding Sites

Keywords

  • eIF5A
  • hypusine
  • polyamine
  • polycistronic vector
  • post-translational modification

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Bioengineering
  • Molecular Biology

Cite this

Production of active recombinant eIF5A : Reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes. / Park, Jong Hwan; Dias, Camila A O; Lee, Seung Bum; Valentini, Sandro R.; Sokabe, Masaaki; Fraser, Christopher S.; Park, Myung Hee.

In: Protein Engineering, Design and Selection, Vol. 24, No. 3, 03.2011, p. 301-309.

Research output: Contribution to journalArticle

Park, Jong Hwan ; Dias, Camila A O ; Lee, Seung Bum ; Valentini, Sandro R. ; Sokabe, Masaaki ; Fraser, Christopher S. ; Park, Myung Hee. / Production of active recombinant eIF5A : Reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes. In: Protein Engineering, Design and Selection. 2011 ; Vol. 24, No. 3. pp. 301-309.
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