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)


    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
    Issue number1-2
    Publication statusPublished - 2005
    MoE publication typeA1 Journal article-refereed


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


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