Verhaert R M, Van Duin J, Quax W J
Pharmaceutical Biology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Biochem J. 1999 Sep 1;342 ( Pt 2)(Pt 2):415-22.
The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.
来自粪产碱杆菌的大型异源二聚体青霉素G酰化酶展示在噬菌体fd的表面。我们将编码序列（α亚基-内部肽-β亚基）与噬菌体外壳蛋白的基因融合。使用修饰的g3p信号序列将多肽引导至周质。在此我们表明，异源二聚体酶可以表达为融合蛋白，该融合蛋白成熟为与噬菌体fd的外壳蛋白相连的活性生物催化剂，从而产生一种噬菌体，其中β亚基共价连接，α亚基非共价附着。该酶可以与次要外壳蛋白g3p融合展示，也可以与主要外壳蛋白g8p融合展示。在这两种情况下，噬菌体上的青霉素G酰化酶与其游离可溶性对应物具有相同的米氏常数，表明展示的酶具有正确的折叠和催化活性。在噬菌体上展示异源二聚体不仅允许其在蛋白质工程中进一步使用，还提供了将该技术应用于将酶分泌到细胞外培养基中的可能性，便于蛋白质的纯化。以青霉素酰化酶为例，进一步探索了噬菌体fd功能性展示蛋白质的上限。尽管使用了为此目的设计的基因构建体，但未观察到多价展示。这些结果结合由g4p多聚体形成的孔径进行了讨论。
