生物工程外文翻譯--flash 和 reash的蛋白標(biāo)記（英文）

1、15Protein Labeling With FlAsH and ReAsHThomas Machleidt, Matt Robers, and George T. HansonSummaryThe ability to image biochemical and phenotypical changes in living cells has become crucial for theinvestigation and under

2、standing of the molecular mechanisms that govern all physiological cellular functions in health and disease. Genetically encoded reporters derived from fluorescent proteins (FPs) have proved to be extremely useful for lo

3、calization and interaction studies in living cells. However, the large size and spec- tral properties of FP impose certain limitations for their use. The recently developed Fluorescein Arsenical Hairpin (FlAsH/tetracyste

4、ine) binder technology emerged as a promising alternative to FP for protein label- ing and cellular localization studies. The combination of a small genetically encoded peptide tag with a small molecule detection reagent

5、 makes this technology particularly suitable for the investigation of biochemical changes in living cells that are difficult to approach with fluorescent proteins as molecular tags. We describe the practical application

6、of this technology to image protein dynamics in living cells.Key Words: Biarsenical; FlAsH; fluorescein; fluorescent proteins; Gateway; high content screening; livecell imaging; Lumio; protein kinase C; protein labeling;

7、 ReAsH; resorufin; tag; tetracysteine.1. IntroductionCurrent high content analysis is mostly performed as end-point analysis using immunofluo-rescence as the primary labeling/detection method. This method although valuab

8、le because of its flexibility and ease of use is by default limited to end-point analysis. The ability to perform real- time analysis of protein dynamics in living cells is critical for the in-depth understanding of the

9、complex biochemical and phenotypical changes associated with cell behavior and function (1). Traditionally, detection and analysis of proteins in living cells relied primarily on the use of flu- orescent proteins (FPs) a

10、s genetically encoded tags (2). Despite its proven versatility and utility for the analysis of protein dynamics, there are certain limitations for the use of FPs. Because of their size (28 kD) FPs have the potential to i

11、nterfere with the activity, localization, or conforma- tion of its fusion partner and can be usually fused only to the N- or C-terminus of a protein (3). In addition, FPs offer only limited spectral variety with a relati

12、ve paucity of useful red FP ver- sions, although the development of improved red FP variants has been reported recently (4).In recent years, a number of alternative live cell labeling methods have been introduced, solv-i

13、ng some of the problems of FPs (such as the spectral limitations) (5,6). However, all published methods rely on the fusion of rather sizable polypeptides to the protein of interest, and, there- fore, share with FP, the d

14、rawback of potential steric interference of the tag with the function of the target protein.209From: Methods in Molecular Biology, vol. 356: High Content Screening: A Powerful Approach to Systems Cell Biology and Drug Di

15、scoveryEdited by: D. L. Taylor, J. R. Haskins, and K. Giuliano © Humana Press, Inc., Totowa, NJThere are currently no examples for the use of FlAsH in high content screening (HCS) appli-cations published in the scie

16、ntific literature. Existing HCS analysis in living cells relies exclu- sively on the use of FP as the tagging/detection method, with the previously mentioned limitations inherent to this approach. The TC/FlAsH labeling t

17、echnology should prove valuable in comple- menting FP in live cell imaging. The combination of small tag size and the high cell permeability and affinity of FlAsH/ReAsH provide the ideal means of pursuing challenging app

18、lications such as multiplex imaging, FRET-based analysis of protein conformation/interaction (10,20,21) and pulse chase experiments (14) in living cells.In this report we attempt to provide a systematic and practical int

19、roduction to the use ofTC/FlAsH technology for the labeling of proteins in living cells. Although staining of TC-tagged proteins in living cells with FlAsH is a relatively simple and straightforward procedure, the reader

20、 should keep in mind that new applications usually require a certain degree of optimization and empirical testing of some of the variables we describe to obtain the desired results. As previously mentioned, TC/FlAsH labe

21、ling has been successfully used for a variety of specialized imaging and biochemical applications (Table 1). A detailed description of these applications would exceed the scope of this report. The reader is instead refer

22、red to the literature for further information.2. Materials2.1. Generation and Expression of TC-Tagged Fusion Proteins1. TC-tagging expression vector (e.g., pCDNA6.2?/Lumio-Dest, Invitrogen, www.invitrogen.com). 2. Reagen

23、ts and equipment for standard molecular biology techniques. 3. Escherichia coli TOP10? (Invitrogen). 4. CHO-K1 cell line (or any other cell line suitable for live cell analysis by microscopy). 5. Standard cell culture re

24、agents and equipment required for the cultivation of mammalian cells (plasticware: Corning, www.corning.com\lifescienes\, all media/supplements: Invitrogen-GIBCO).Protein Labeling With FlAsH and ReAsH 211Table 1 Publishe

25、d FlAsH ApplicationsMethod Protein of interest Cell system ReferenceFluorescence assisted Synaptotagmin connexin 43 Drosophila, third 9,11light inactivation instar larvaeFRET A2A adenosine receptor CHO 10Conformation

26、al analysis – Calmodulin HEK293 12,13,26environment sensitive probeElectron microscopy Connexin43 HeLa 14,15Single molecule Calmodulin n.a. 16Detection/analysisAffinity purification Kinesin n.a. 17Protein translocation

27、/tracking Connexin43 HeLa 14,15HIV HeLa, Jurkat 18Glucocorticoid receptor Hela, CHO Fig. 3APKC-α HeLa, CHO Fig. 3Bβ-tubulin HeLa, CHO Fig. 3Cβ-tubulin Saccharomyces 2Cytochrome-c cerevisae 19Multicolor pulse chase analys