Wehrman Tom, Kleaveland Benjamin, Her Jeng-Horng, Balint Robert F, Blau Helen M
Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3469-74. doi: 10.1073/pnas.062043699.
We have defined inactive alpha and omega fragments of beta-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the alpha fragment increased complemented enzyme activity by up to 4 orders of magnitude. beta-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.
我们已经定义了β-内酰胺酶的无活性α片段和ω片段，它们在细菌和哺乳动物细胞中都能互补形成一种功能性酶，作为与这些片段融合的蛋白质之间相互作用的一种读出方式。这一进展的关键在于鉴定出一种三肽，天冬酰胺-甘氨酸-精氨酸，当它并列于α片段的羧基末端时，可使互补酶活性提高多达4个数量级。β-内酰胺酶非常适合监测组成型和诱导型蛋白质相互作用，因为它体积小（29 kDa）、为单体，并且可用一种可透过细胞的荧光底物进行检测。在迄今为止发表的哺乳动物蛋白质相互作用检测系统中，其可忽略不计的背景、酶促放大引起的诱导信号强度以及数分钟内即可检测到信号，都是无与伦比的。
