An exploration of GFP.pdf

An exploration of GFP, its spectral properties and its applications in Cell Biology

ABSTRACT

Nowadays there is a growing interest in the search for new biomarkers. These can be defined as small molecules, proteins or enzymes that can be measured and employed for diagnostic and /or therapeutic purposes. Also they can serve as an indicator of a physiological or pathological process or of the exposure to certain toxic agents. Proteins that are being used more nowadays as biomarkers are proteins with the property of emitting green light like the Green Fluorescent Protein (GFP) produced by the bioluminescent jellyfish

 Aequorea

victoria . In these practices, we analyze the fluorescence properties of a modified form of GFP,

called Emerald GFP (EmGFP) contained as the gen of interest in a protein expression vector of a plasmid, which we will use to transform colonies of  E.

coli.

Then we will induce the

expression of EmGFP with IPTG and purify by metal affinity immoblised chromatography (IMAC).Finally we analyze the use of fluorescent markers to study and monitor the cellular process of phagocytosis in cells that participate in the immune system of insects. To do this we injected cells of  E.  E. coli transformed with the EmGFP and other fluorescent marker called Fluorescein isothiocyanate (FITC) in the abdomen of  Drosophila  Drosophila melanogaster  melanogaster  specimens so we can then extract and analyze their haemolymph. In our experiment we observe that it is effective to use GFP to monitor the process of phagocytosis in insect cells. This is important because it allows us to check the insect immune response against invasive agents and their effectiveness. The study of the immune response in different animal species may open new avenues to combat infections in humans.

KEYWORDS AND ABBREVIATIONS 

GFP - Green Fluorescent Protein



EmGFP - Emerald Green Fluorescent Protein



FITC - Fluorescein isothiocyanate



 E. coli -  Escherichia coli



IMAC - Metal Affinity Immoblised Chromatography



nm - nanometers



DAPI - 4',6-diamidino-2-phenylindole



 D. melanogaster - Drosophila melanogaster 

INTRODUCTION

A biological marker is a characteristic or a physiological, biochemical or morphological change that it´s objectively measurable in a molecular, biochemical or cell level, and acts as an indicator of biological normal or pathological processes. The analytical and chemical characteristics that should have the markers are: • specificity and sensitivity; • ease of sampling (avoid, where possible, the use of 

invasive techniques) and

representativeness; • formation kinetics well known and

established;

• stability.

In general it can be said that the most common analytical tests to detect markers in biological samples are grouped into three general types of tests: biological, physicochemical and immunological. Immunological techniques have high sensitivity and selectivity and are based on antigen-antibody and /or enzyme-substrate reactions, allowing studying a large number of  molecules. Selectivity is a very important feature in the experiments, because by this way you avoid

erroneous

results (false

positives

and false

negatives).

It

can

be

used radioimmunoassay (RIA) as an immunological technique with great clinical application, detecting antigens or antibodies in biological fluids using some radioisotopes as markers (H3, C14 y I125). We can also use plasmids to insert protein expression vectors containing the gene coding for the marker we analyze, in our case EmGFP. The cellular processes which can be explored using labelling methods are: (i) Cell identification following electrophysiological recording. (ii) Delineation of cellular architecture in anatomical studies. (iii) Tracing neuronal pathways. (iv) Identification of cell progeny in lineage studies. (v) Investigations of the transfer of molecules from one cell to another via gap junctions or other routes.

(vi) The introduction of genetic material that affect protein synthesis or gene expression. (vii) The measurement of intracellular ion concentrations, for example pH or calcium ions. In our experiment we analyze the use of fluorescent proteins, specifically EmGFP as a marker to study and monitor the cellular process of phagocytosis in cells that participate in the immune system of insects. Fluorescence is the emission of electromagnetic radiation in the visible region by a molecule or atom after the initial absorption of a photon. The discovery of fluorescent proteins of native aquatic organisms that possess bioluminescence has been a revolution in cell biology. There are natural fluorescent proteins that emit fluorescence in the range of  different colours, like green and red. The protein from the jellyfish Aequorea victoria, which they called native Green Fluorescent Protein (GFP), has been the biggest impact in the scientific community. The green fluorescent protein has two absorption peaks at 395 and 475 nm. When illuminated with UV light, GFP displays a major absorption peak at 475 nm, resulting in fluorescence at 310 nm. When GFP is not illuminated with UV light, it displays a major absorption peak at 395 nm. The tertiary structure of the protein consists of a beta barrel of 40 Å, formed by eleven antiparallel β leaves, accompanied by α helix whit the chromophore in the center of the structure. The GFP has a chromophore p-hidroxibenzilideneimidazolinon subject to the helix within the barrel. This chromophore is formed by spontaneous cyclization and oxidation of residues 65-67, corresponding to amino acids Ser 65 - Tyr 66 - Gly 67 of the native protein and is responsible for the emission of green light. We knows dozens of mutated proteins derived from GFP of  Aequorea

victoria

(native), whose alterations cause different physicochemical properties to the original, and in which their expression is enhanced (increased fluorescence, changes in the range of excitation and emission ,photostability, etc.), but without changing the overall behaviour of the protein. In our practices we used a modified form of GFP, called Emerald GFP (EmGFP). The fluorescence properties of EmGFP differ slightly to the wild type GFP and we shall monitor the EmGFP by excitation at 487 nm and detection at 509 nm. In these practices we also used the FITC as tool to monitor the process of phagocytosis in insect cells. FITC is an amine-reactive derived from the fluorescein that has a wide ranging application as an antibody labels and as other testing labels for the use in fluorescence

microscopy, flow cytometry and immunofluorescence-based assays. FITC has an excitation spectrum peak at wavelengths of approximately 495 nm and an emission spectrum peak at wavelengths of 521 nm. Insects, such as organisms that interact in different environments and ecosystems are exposed to the attack by the insects themselves, in the case of natural enemies such as parasitoids, and a large number of pathogens microorganisms with infectious characteristics such as fungi, bacteria, viruses, nematodes, protozoa and riketsias; against which the insect requires defense, either using their own innate resources or developing defense mechanisms. The insect immune reactions usually occur when a foreign body is introduced into their system. Reactions presented in the hemolymph as cellular and humoral defense can reduce or eliminate the effect of invading body. The most important cellular defense reactions include phagocytosis and encapsulation of foreign bodies. Free cells of the haemolymph called hemocytes are the effectors of these reactions. They fail to control humoral factors contained in plasma or serum, humoral factors may enhance or reduce the cellular defense reactions. The phagocytic process generally involves the following steps: (1) Recognition by the hemocytes of foreign particles within the insect hemocoel. (2) Chemotactic attraction where hemocytes are attracted to foreign particles. (3) Adhesion occurs when the particles adhere to the surface of the hemocytes. Here are physicochemical forces involved in the receptor for molecules of the foreign particles, establishing itself binding sites with the surface of the hemocytes. (4) Ingestion of the particle occurs when the pseudopodia or membrane invaginates around the particle and seals it. In the cytoplasm, the phagocytic vacuole membrane comes from the original phagocytic cell membrane. (5) Degranulation is the released of enzymes from the granules (lysosomes) within the phagocytic vacuole. (6) Lysosomal digestion of particles occurs by enzymes and undigested particles are expelled by exocytosis.

METHODS

Week 1: Preparation of BL21 (DE3) competent cells and transformation with pRSET/EmGFP expression plasmid. Competent cells, named this way because of the physiological state in which these cells are, one in which they can take genetic material from the surrounding environment. Logically the genetic material will be one of our interests for the transformation experiment, the pRSET/EmGFP. 1. At first we where given a diluted culture of  E. Coli BL21 (DE3) that had grown the night before in an LB medium. 2. Then we made the cell transformation using a thermal shock technique with CaCl2, a type of salt that increases the transformation efficiency. The steps of the technique are to cool º

down the cells (4 C), so we make them competent cells for the technique; and then give them º

a fast heat shock (42 C), so that the plasmid flows inside the opened cell pores into the bacterial cytoplasm. 3. Finally we resuspend the cells on LB liquid medium with Ampicillin and putted them in a º

growing plate to incubate overnight at 37 C. 4. The cells that should grow in this medium with the antibiotic are the transformed cells because they have the resistant gene against the Ampicillin, the selection marker contained in the pRSET plasmid.

Week 2: Induction of GFP over expression in the

 E. coli

previously transformed.

-  E. Coli BL21(DE3), it is characterized for many factors, some of wich are: reaching a high optical density, is not pathogenic and it´s deficient on ompT and Lon expression ( two extracellular and intracellular proteases respectively) that can degrade the protein of interest. This strain also has a sequence encoding for the T7 polymerase of RNA which depends on the lacUV5 and the expression of the LacI repressor (elements from the lac operon). This is the reason why the expression vector (with GFP) used to transform this strain has a recognition sequence for this polymerase (pT7 of the pRSET) of high rate transcription. 1.In these practice, first we where given the transformed bacterial culture in a state of  exponential growth to optimizes the growing and visualization of our culture after the induction. 2.

Afterwards

we

induced

the

GFP

expression

using

IPTG.

- IPTG or Isopropyl β-D-1-thiogalactopyranoside, a non-hydrolyzable analog of allolactose (hidrolyzable by the cell), wich will activate the transcription of the protein expression vector (GFP), but unlike the allolactose, in a controlled way. The allolactose and IPTG induce the transcription of the lac operon because they join to the LacI repressor, so is very common to use regulatory elements from the lac operon as inducible systems for the protein expression. In our experiment, the IPTG will join to the lacI repressor so that the T7 polymerase can be transcribed so this one can after transcribe the GFP in our plasmid. 3. Finally, after an incubation and before we harvested the cells and froze them for the storage; we checked if the bacterial where producing GFP using a fluorescence microscopy.

Week 3: Purification and analysis of GFP. Our expression vector generates a tail of histidine (His6) next to the GFP that will act as a “tag” that will simplify the purification process by affinity chromatography. However, this tag

can affect the tridimensional structure and sometimes the digestion at the specifics sites of the protein; these are some reasons why the pufication in the end it´s not valued as 100%. 1. We first homogenized the bacterial culture from last week using some products such as benzamidine, lysozyme, Triton X-100, DNase I and Rnase A. Out of wich none will affect to the structure or function of the GFP. 2. After we passed on to the metal affinity chromatography (IMAC). - Metal affinity chromatography (IMAC). Where we use a chromatography column containing a Ni-NTA (Ni 2+-nitriloacetic acid) agarose matrix, wich has positive charges in the metal ion (Ni 2+) that will attract the negative ions from the nitrogen in the histidine residues that act as the “tag” for our interest protein. The imidazole has practically the same

structure as the R´ group if the His aminoacid; so we used this molecule in the elution procedure with increasing concentrations between 20mM and 250mM to displace the GFP(His6) from the Ni-NTA for eluting and separating the rest of the homogenate from our purified protein of interest. 3. Finally we analyzed and determine the amount of GFP present in the elution tubes using a spectrophotometer that measured the absorbance.

Week 4: Use of GFP in monitoring phagocytic activity of haemocytes in adult haemolymph. In the last week we used GFP to monitor the phagocytic activity of hemocytes of the hemolymph of an adult specimen of  D. melanogaster . 1. First, adult flies were anesthetized with CO2 and injected with cell transformants of  E. coli expressing the EmGFP or FITC. This step of the practice had been developed previously. 2. After this, we extracted the contents of the abdomen of the flies and we put them into a buffer. We let the cells resting for a few minutes to allow the fat droplets from the hemolymph go to the top of the surface and thereby separate the blood cells of them. The last week we used GFP to monitor the phagocytic activity of hemocytes of the hemolymph of  an adult specimen of  D. melanogaster . 3. Finally we put a small amount of our sample on a slide and added a drop of VectaShield R containing DAPI, which allows to view the nucleus of the cells. Then we covered it with a cover slip and kept it in darkness to avoid the photooxidation before we observe the samples under the Axiovert 135 microscope, using the appropriate filters.

RESULTS

After making purification of the EmGFP we measure the absorbance at 488 nm of each fraction eluted to determine the amount of GFP present in our samples. To do this we used the Absorbance Coefficient of EmGFP at 488nm and the following formula. 

Absorbance Coefficient of EmGFP at 488nm is 53, 000 M-1 cm-1.



Formula: A=ExC,

where

A=absorbance,

E=the absorbance coefficient and

C=concentration So we present our results in the following table: Absorbance

Concentration of GFP

0,021

3,96x10 M

0,014

2,64x10 M

0,049

9,25x10 M

0,045

8,49x10 M

0,069

1,30x10 M

0,027

5,09x10 M

0,01

1,89x10 M

0,012

2,26x10 M

-7 -7 -7 -7 -6 -7 -7 -7

After extraction of the hemolymph of the flies and preparation of samples, we observed them in the Axiovert 135 microscope using appropriate filter sets. The images obtained were as follows: Figure 1(a) FITC labelled E. coli phagocytosis

Image 1(b) FITC labelled E. coli phagocytosis (DAPI staining)

Image 3(a) GFP labelled E. coli phagocytosis

Image 3(b) GFP labelled E. coli phagocytosis (DAPI staining)

DISCUSSION

Respect to

the

purification

of GFP we

used IMAC, for

which was

used a resin

containing metal ions Ni2+ which has high affinity for the imidazole rings of histidines tails that

present our GFP. The elution procedure was

performed with

concentrations

of imidazole between 20mM

and 250mM. We

concentrations

of GFP were

the eluent tubes for 50mM

obtained

in

note

increasing

that the and

highest 100mM

concentrations of imidazole so we assume that the purification was successful. We could see perfectly, thanks to the labelling with GFP and FITC, E. coli cells that had been phagocytized. This was possible thanks to the use of DAPI, which allows the observation of  the nucleus of hemocytes because it appears stained in blue. We could observed this, we use different filters in the microscopy that allow the passage of specific wavelengths that correspond to the GFP or the FITC.

CONCLUSION

The use of labelling techniques with fluorescent proteins is an accessible and useful tool for the study the different processes that happen inside of many organisms. This is important because it allows us to understand better the physiology of all the different species present in our world, and it can also open doors to new sciences. This techniques are useful too for the early detection of different diseases such as neurodegenerative diseases, cardiovascular diseases and tumors.

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