Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage

Eeva-Christine Brockmann (Corresponding Author), Urpo Lamminmäki, Petri Saviranta

Research output: Contribution to journalArticleScientificpeer-review

3 Citations (Scopus)

Abstract

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.

Original languageEnglish
Pages (from-to)146 - 154
Number of pages9
JournalBiochimica et Biophysica Acta: General Subjects
Volume1724
Issue number1-2
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

Fingerprint

Dihydropteroate Synthase
Bacteriophage M13
Bacteriophages
Display devices
Cysteine
Protein Engineering
Mutagenesis
Mutation
Proteins
Disulfides
Libraries
Conformations

Keywords

  • Dihydropteroate synthase
  • Phage display
  • Protein expression
  • Protein engineering
  • Cysteine residue

Cite this

Brockmann, Eeva-Christine ; Lamminmäki, Urpo ; Saviranta, Petri. / Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage. In: Biochimica et Biophysica Acta: General Subjects. 2005 ; Vol. 1724, No. 1-2. pp. 146 - 154.
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Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage. / Brockmann, Eeva-Christine (Corresponding Author); Lamminmäki, Urpo; Saviranta, Petri.

In: Biochimica et Biophysica Acta: General Subjects, Vol. 1724, No. 1-2, 2005, p. 146 - 154.

Research output: Contribution to journalArticleScientificpeer-review

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