Peters Verena, Rehm Bernd H A
Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
J Biotechnol. 2008 Apr 30;134(3-4):266-74. doi: 10.1016/j.jbiotec.2008.02.006. Epub 2008 Feb 17.
Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.
大肠杆菌经过基因工程改造，可在细胞内制造链霉亲和素珠。链霉亲和素的变体（单体、核心和成熟全长链霉亲和素）在C末端与来自食酸戴尔福特菌的聚酯颗粒形成酶PhaC融合。所有链霉亲和素融合蛋白都介导了大肠杆菌中相应颗粒的形成，并在颗粒表面过量表达。单体链霉亲和素只有在作为单链二聚体融合时才显示生物素结合能力（0.7 ng生物素/μg珠蛋白）。核心链霉亲和素和相应的单链二聚体分别介导约3.9和1.5 ng生物素/μg珠蛋白的生物素结合。然而，当使用成熟全长链霉亲和素时，可获得约61 ng生物素/μg珠蛋白的生物素结合能力，平衡解离常数（KD）约为4×10⁻⁸M。使用酶联免疫吸附测定（ELISA）、竞争性ELISA和荧光激活细胞分选（FACS）对展示成熟全长链霉亲和素的珠子进行了详细表征。将生物素化的酶或抗体固定到珠子上以及纯化生物素化的DNA，以证明这些新型链霉亲和素珠的适用性。本研究提出了一种利用工程化大肠杆菌作为细胞工厂，廉价高效地一步生产通用链霉亲和素珠的新方法。
