Bimolecular Fluorescence Complementation and Immunocytochemistry of Pap

Lab 02

Bimolecular Fluorescence Complementation and Immunocytochemistry of PAP

Name: Mina Mohammed-Ali

Partner: Wasan Fakhri

Student number: 211547312

T. As: Varvara and Paul

Date: Feb.26th.2016

Introduction:

   Protein-protein interaction is a very powerful biochemical event that revels information about the profile of two specific proteins in question. There are different ways to examine this phenomena, one of which is Bimolecular fluorescence complementation (BiFC), which was used in this lab to observe the transformation (if any) of the Agrobacterium culture carrying the fusion constructs, which was already fused with c-terminus of the pokeweed antiviral protein, into the Tobacco benthamiana plant. Protein-protein interaction can be detected biochemically using wide range of approaches such as, yeast two hybrid system, co-immunoprecipitation pulldown assays, and chemical crosslinking; also using biophysical methods such as Microscale Thermophoresis (MT), Fluorescence resonance energy transfer (FRET), and NMR [1]. Each method has its advantages and disadvantages, in the previous lab experiment we used the yeast two hybrid assay to investigate if two proteins interact together, which is a good system to test interactions in vivo, but it relies on the distance separating the two proteins in question, and the presence of a functional activation and binding domains, in addition to the time it requires to assure that there are no off site interactions. This limits the visualization to only specific area of the genome; moreover, it requires multiple treatments to make sure that there is no external factor binding to the domains and causes a false-positive results, and false-negative, which are considered the major drawback of this technique. On the other hand, using fluorescent expression of proteins (BiFC) could allow boarder spectrum of investigation and give more assured results, since the interaction is visualized by the expression of a green fluorescent in living cells; in addition, it dose not require the amount of labour that is involved in other methods, and it is faster compared to yeast-two hybrid system [2].

[pic 1]

Figure (1): an illustrative figure showing the two proteins of interest when tagged with one half of the fluorescent protein and how when they come together a green fluorescent is detected [3].

  BiFC approach is particularly vital in determining the locations of the subcellular protein interactions, when two complement segments of the proteins that are attached to a half of fluorescent molecule, and only when they two segments come together fluorescent protein is expressed [4]. This technique was employed to visualize the interaction between two non fluorescent protein fragments of the pokeweed antiviral protein (PAP) that were tagged with half of the full fluorescent protein on each, and examined using confocal microscopy. Agrobacterium cultures were already transformed with constructs of the antiviral protein of the pokeweed that was attached to the fluorescent protein form the N-terminus. These cultures were used to make a transgenic tobacco and using the confocal microscope we were able to investigate the presence or absence of a YFP expression.

  One of the plants used was Nicotiana benthamiana, which belongs to the Solanaceae family, and it is considered the most preferred organism in tissue culture and other plant biology studies for various reasons, one of which is there is well known information regarding its behavior towards different pathogens; moreover, its genomic sequence is known [5]. A study that was done by Walter and colleagues in 2004, suggested that upon successful transfection of the Agrobacterium constructs into the Nicotiana benthamiana leaf’s cells, the effectiveness of obtained results depended on the type of constructs used [6].

The other plant is commonly called pokeweed which is known scientifically by Phytolacca Americana, and it belongs to the Phytolaccaceae family, and it contains an antiviral protein that is known for its toxicity to the mammalian cells, which functions by inhibiting the ribosomal machinery [7]. 

  Confocal microscopy is a very common tool that is used in biology to identify when two proteins come together forming a full yellow fluorescent protein (YFP). It functions by increasing the optical resolution such that it only shows what is in focus; while eliminating anything out of focus, which will appear in black. The presence of a pinhole in the microscope, had the advantage of directing the light to only what is in the focus, and shedding the light on a thin section of the specimen in question, which can use multiple images to create a three dimensional view of the tissue [8].

  Protein interactions form a network of events that influence many cellular aspects in the body. Proteins interaction could yield different functions in different tissue types. Visualizing the location of interaction reveals information about their regulation profile and how they function inside the cell. This can be observed using a technique called immunocytochemistry that employs the specificity of binding of a primary antibody, which is recognized by a secondary antibody to detect the location of proteins in action, and all this can be visualized using fluorescence microscope [9].

  This lab report examines the ability of the Nicotiana benthamiana (Tobacco) to uptake a transgene from an Agrobacterium culture containing a pokeweed antiviral protein (PAP) fused with a fluorescent protein. In addition to investigating the profile action of a wild type of the protein and the mutant using BiCF. Moreover, employing immunocytochemistry to visualize the location of proteins expressed endogenously and exogenously using pokeweed and toPAPcco respectively. The hypothesis is, upon transduction of the tobacco plant with the Agrobacterium, which is carrying the YFP-protein constructs, if successful, the two proteins interact and a green light will be detected to a black background when examining the samples under the microscope. Besides, the location of the protein expression will be different in toPAPcco and transgenic tobacco.