Purification of Recombinant Gfp Produced by Agrobacterum Mediated Transient

نویسنده

  • N. V. KUCHUK
چکیده

16 Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium mediated transient expres sion in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and included ammonium sulfate precipitation and Q sepharose anion exchange chromatogra phy. It results in obtaining of a fraction with about 85 % GFP homogeneity and the protein yield of about 75 %. Introduction. During the recent decade green fluorescent protein (GFP) has become one of the most popular in vivo marker molecule used in great variety of biological and medical experiments, e.g. researches on recombinant protein expression in different cell systems, studies on promoter activi ties, protein targeting, localization, kinetics and functional analysis of cytoskeleton and cytoskele ton associated proteins etc [1, 2]. At the present time GFP gene can be regarded as a substitution of β glucuronidase gene, which is widely used as a reporter (gene) in transformed plants. In contrast to GUS gene product, GFP can be directly moni tored or quantified in living cells without destruc tive tests. GFP is derived from jellyfish Aequorea victoria and contains a chromophore which does not require any substrates or cofactors for fluorescence. After cloning GFP has undergone substantial mod ifications that resulted in high expression rate, increased fluorescence, stability and low toxicity for a wide range of hosts, including plant cells [3–5]. The present GFP based protocols include qualitative and quantitative analyses of GFP expression by detection of its fluorescence from the cellular level to the whole plant level [1, 2]. For cor rect quantitation considerable amount of purified GFP is necessary for building of calibration curve. Transient expression of foreign genes is a recently developed method allowing production of large amount of recombinant proteins within a very short (days) time [6]. It occurs without stable inte gration of foreign DNA into the host genome. Our recent work describes optimization of protocol for rapid and high scale production of recombinant GFP using transient expression in Nicotiana species [7]. Although numerous publications exist con cerning recombinant GFP production and purifi cation, most of them describe purification of GFP from bacterial source. It includes tag based proto cols [8, 9], which not always allow to separate improperly folded or cyclized GFP from correctly folded form of this protein [10], as well as size exclusion chromatography [11], chromatofocus ing [12], organic extraction [11] etc. On the other hand, GFP production and purification from plant source under physiological conditions using ion exchange chromatography may be regarded as an alternative method (including GFP purification from constitutively transformed plant tissue). In the present work we describe a scheme including УДК 57.085.2 + 582.926.2 + 577.21 Y.R. SINDAROVSKA, Y.V. SHELUDKO, I.M. GERASYMENKO, M.A. BANNIKOVA, N.V. KUCHUK Institute of Cell Biology and Genetic Engineering, Zabolotnogo str. 148, Kyiv 03680, Ukraine E mail: [email protected]

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تاریخ انتشار 2012