Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain

Andrew Hayhurst

    Research output: Contribution to journalArticle

    51 Scopus citations

    Abstract

    The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis. Single-chain antibody fragments (scAb) are scFv with a human κ light chain constant (HuCκ) domain attached C-terminally and share similar problems of expression. By fusing the E. coli maltose-binding protein (mbp) gene either 3' or 5' to a scab specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion. The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely. Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scab in the periplasmic compartment, indicating that periplasmic scab solubility is unrelated to propensity to cause growth arrest. IMAC purification of scab and mbp-scAb proteins followed by liquid competition ELISA revealed the IC50 for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding. The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCκ domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion. The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCκ domain fusion were extended to a second scFv specific for the herbicide diuron. Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCκ) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E. coli periplasm. (C) 2000 Academic Press.

    Original languageEnglish (US)
    Pages (from-to)1-10
    Number of pages10
    JournalProtein Expression and Purification
    Volume18
    Issue number1
    DOIs
    StatePublished - Feb 2000

    Keywords

    • Atrazine
    • Maltose-binding protein
    • Protein solubility
    • Single-chain antibody fragment
    • Translocation

    ASJC Scopus subject areas

    • Biotechnology

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