Purification and characterization of human-derived Pneumocystis jirovecii dihydrofolate reductase expressed in Sf21 insect cells and in Escherichia coli

Vivian Cody, Kim Chisum, Carleen Pope, Sherry Queener

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Pneumonia caused by Pneumocystis jirovecii is still a major opportunistic infection among patients with AIDS. This opportunitistic pathogen is susceptible to therapy with inhibitors of the enzyme dihydrofolate reductase (DHFR) that target cell growth. Recent studies have shown that recombinant human-derived Pneumocystis DHFR (pDHFR) differs from rat-derived pDHFR by 38% in amino acid sequence. However, characterization of drug susceptibility, kinetics, and the three-dimensional structure of human-derived pDHFR has been hampered by the limited availability of purified material. The present study was undertaken to develop procedures to prepare sufficient enzyme for structure/function studies. Protein yield was limited when human-derived pDHFR was expressed in Escherichia coli using a pET28a(+) vector with an N-terminal His-tag for the 25 kDa protein. Therefore, the protein was expressed in Sf21 insect cells by baculovirus infection. The soluble enzyme was purified from cell lysates via Ni-chelated chromatographic columns, yielding about 5.1 mg of human-derived pDHFR fusion protein per liter of Sf21 culture. The purified protein had the expected mass as determined from Western blots with antibody for the N-terminal His-tag. This His-tagged recombinant DHFR from human-derived Pneumocystis was catalytically active and demonstrated kinetics similar to the recombinant enzyme from rat-derived Pneumocystis. The present studies for production of soluble human-derived pDHFR indicated that the baculovirus expression system supported production of significantly purer catalytically active enzyme in higher yields than that expressed in bacterial cultures. These protocols now make it possible to facilitate screening of antifolates with selectivity for human-derived pDHFR and to determine its three-dimensional structure.

Original languageEnglish
Pages (from-to)417-423
Number of pages7
JournalProtein Expression and Purification
Volume40
Issue number2
DOIs
StatePublished - Apr 2005

Fingerprint

Pneumocystis
Sf9 Cells
Pneumocystis carinii
Tetrahydrofolate Dehydrogenase
Escherichia coli
Purification
Insects
Enzymes
Proteins
Rats
Folic Acid Antagonists
Baculoviridae
Kinetics
Cell growth
Enzyme Inhibitors
Pathogens
Screening
Fusion reactions
Availability
Amino Acids

Keywords

  • Antifolate inhibitors
  • Baculovirus
  • Recombinant hpDHFR

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of human-derived Pneumocystis jirovecii dihydrofolate reductase expressed in Sf21 insect cells and in Escherichia coli. / Cody, Vivian; Chisum, Kim; Pope, Carleen; Queener, Sherry.

In: Protein Expression and Purification, Vol. 40, No. 2, 04.2005, p. 417-423.

Research output: Contribution to journalArticle

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