Hey guys,

I thought you might like to see some of the stuff I do in school. this is the essay I worked on with Cindy (dunt, dunt, daaaaa!) This is an incomplete version (no figure headings, probably some crappy writing left over from you know who) anyway, I'm sure you'll find it boring, but this is my excitement!!

P.S. the pictures didn't copy for some reason.

The Cleavage Mechanism, Production, Purification and Characterization of a Type II Restriction Endonuclease, Eco R1

Restriction enzymes are found almost exclusively in bacterial cells. These enzymes recognize unique pallindromic DNA sequences approximately 4-8 base pairs in length and cut the DNA within or in close proximity to the recognition site (Pingoud and Jeltsch, 2001). Linn and Arber (1968), suggest that restriction enzymes have evolved in prokaryotic bacterial cells as a defense mechanism that enable the host to degrade bacteriophage DNA that may infect and cause harm to the cell. The products of restriction enzyme cleavage are DNA fragments containing 5’ phosphate and 3’ OH ends that are not harmful to the host cell and may exist as blunt ends, 3’ overhangs or 5’ overhangs depending on the nature of the restriction enzyme (Fig 1 p493 Alberts et al.).
1.a. blunt ends

b. staggered ends

There are two general types of restriction enzymes known: restriction exonucleases that cleave DNA from exposed 5’ and 3’ ends and restriction endonucleases that cleave within the DNA strand at specific pallindromic sequences. These categories can be further broken down to sub-categories directed by molecular structure, and cleavage mechanisms. However, this essay will focus specifically on the structure, mechanism, purification, production and characterization of type II restriction endonucleases (i.e. EcoR1) which are defined as restriction enzymes that cleave within or in close proximately to the pallindromic recognition sequence and do not require ATP hydrolysis for its catalytic function (Pingoud and Jeltsch 2001).
Although the molecular structure of restriction endonucleases is variable amongst the enzymes, all restriction endonucleases are known to consist of two or more polypeptide chains and a common core composed of five β-sheets flanked by α-helices (Pingoud and Jeltsch 2001). An illustration of this structure is present in Figure 2 which displays the molecular structure of type II restriction endonuclease EcoR1.

(Figure 2 will go here, I couldn’t cut and paste it yet but I’ll either attach the figure to this email or show you in class)

A catalytic centre responsible for DNA cleavage is located in the common core of restriction endonucleases and contains two carboxylates (typically asparate or glutamate) that bind to Mg2+ or another cation which decreases electrostatic repulsion that exists between the protein and DNA (Pingoud and Jeltsch 2001).
While unattached to DNA, the restriction endonuclease exists in a non-specific conformation with a secondary structure that isolates the catalytic centre from surrounding molecules to prevent undesired reactivity within the catalytic site (Pingoud and Jeltsh 2001). The mechanism of cleavage begins with the non-specific binding of the restriction enzyme to the DNA. In this first contact, the endonuclease is loosely bound to the DNA by hydrogen bonds and the catalytic centre remains quite a distance away from the DNA (Pingoud and Jeltsh 2001). The enzyme will then progress along the DNA for a short distance. This movement is known as one-dimensional diffusion and includes sliding, hopping and intersegmental transfer of the restriction endonuclease (Pingoud and Jeltsh 2001). In EcoR1, it has been shown that sliding is the most important process of restriction site recognition. If the recognition sequence is present within the short distance surveyed by the restriction endonuclease, further conformational changes of the DNA and restriction endonuclease occur to activate the catalytic centre that cleaves the phosphodiester bond connecting two DNA bases (Pingoud and Jeltsh 2001). Cleavage is also accompanied by the release of counterions surrounding the complex and the partial dehydration of the protein (Pingoud and Jeltsch 2001). Following the cleavage of the phosphodiester bond, the DNA fragments and enzyme are released either by direct dissociation of the enzyme-product complex or by the transfer of the enzyme to non-specific areas of the same DNA (Pingoud and Jeltsh 2001). For EcoR1, linear diffusion is known to accelerate the process of target site location and dissociation following phosphodiester bond cleavage (Pingoud and Jeltsh 2001).
Pingoud and Jeltsh (2001) recognize that restriction endonucleases that produce blunt ends or 3’ overhangs typically approach the DNA by the minor groove side, while enzymes that produce 5’ overhangs approach the DNA by the major groove side. This behaviour is correlated to the molecular structure of the enzyme, which dictates it’s ability to form a dimer with the DNA and expose its catalytic site (Pingoud and Jeltsch 2001). As an example, EcoR1 approaches DNA from the major groove side and produces 5’ overhangs.
To prevent the degradation of the host cell’s genomic DNA, prokaryotes have developed a modification system that methylates genomic base pairs associated with the recognition sequence of the endogenous restriction enzyme (Lederberg 1965). In doing this, the restriction endonuclease is unable to detect cleavage sites within the genomic DNA thereby preserving the prokaryote’s genetic information. This modifying technique is also administered to phages or plasmids whose function benefits the host cell (Lederberg 1965).
Type II restriction endonucleases have proven to be very useful in the investigation of protein function, gene sequencing, DNA mapping, mutation identification, recombination, etc. (Vogel et al. 1978). The diversity of restriction enzymes, each of which recognizes a unique DNA sequence, also provides the tools to compose DNA libraries and to expand applications of enzymes in research. To date, more than 3000 type II endonucleases have been identified (Pingoud and Jeltsch 2001) and many of these have been used to supplement more complex or expensive techniques formerly used in DNA, RNA, or protein analysis (Danna and Nathans 1971). For example, coupling restriction enzyme digests with gel electrophoresis has provided a new method for detecting DNA mutations and/or differences within the sequence of DNA fragments (Danna and Nathans 1971). Thus, restriction endonucleases have proven to be a valuable asset to the accessibility, efficiency and accuracy of molecular analysis. Therefore, their production is in high demand within the scientific community for these purposes.
Market Size????
Production Methods
EcoR1 is naturally produced in E. coli cells but at levels too low for commercial production. To increase the expression and yield of EcoR1 from E. coli, Tamerlar et al. (2001) genetically modified two strains of the E. coli bacteria to overproduce the EcoR1 enzyme.
Tamerler et al. (2001) successfully induced an increased production of EcoR1 in E. coli by transforming the bacterial cells with plasmids containing the EcoR1 gene. Plasmids are small, circular double stranded DNA molecules that exist separately from the chromosomal genome of E. coli, and are commonly used to transport desired DNA fragments into a host cell for the purposes of DNA amplification (Ninfa & Ballou, 1998). To do this, the gene of interest is isolated from its natural genome and inserted into the plasmid vector. Unique restriction enzymes create complementary 5’ and 3’ ends on both the desired gene (in this case EcoR1) and the plasmid DNA. The gene is then spliced into the plasmid DNA using DNA ligases to form the recombinant plasmid. The manipulation of gene expression using plasmids with a DNA insert is deemed “recombination” (Ninfa & Ballou, 1998). Once the plasmid containing EcoR1 is transformed into competent host cells, the plasmid DNA replicates by means of its natural method of replication. As the plasmid synthesizes its own DNA, the inserted EcoR1 gene is also replicated. The result is the increased expression of EcoR1 within the host cell thereby increasing the intracellular concentration of the EcoR1 protein. Tamerler et al., 2001 used the strain M5248 carrying the plasmid pSCC2, and strain 294 carrying the plasmid pPG430. They found that E. coli 294 produced the highest yield of enzyme when induced by IPTG, producing 3.3x106 U/g cells compared to 1.3x105 U/g by pPG430 (Tamerler et al., 2001). The techniques ascribed to the process of creating a genetically modified cell will be explained in greater detail in the following sections.
Isolation of Genomic DNA
Donor cells that naturally produce EcoR1 are grown and harvested in the laboratory. The EcoR1 gene is isolated from these cells and used to create the recombinant plasmid. To isolate the EcoR1 gene, the donor cell membranes are degraded using lysozyme enzyme, which degrades polysaccharides in the cell wall of bacteria. The enzyme catalyses the insertion of a water molecule (hydrolysis) at the  1-4 glycosidic bond between monosaccharides, breaking the polysaccharide chain and the cell wall. A detergent, commonly sodium dodecyl sulfate (SDS), is used to solubilize the lipids of the cell wall, thereby removing the cell wall from the solution. The solution is then treated with phenol:chloroform:isoamyl alcohol to extract the DNA from the other cellular components in the solution. Centrifugation of the liquid separates the genomic DNA to the upper aqueous layer of the supernatent. This layer is removed and the DNA is precipitated with ethanol. The genomic DNA collected by centrifugation and resuspended in a buffer solution. It is at this point that the genomic DNA is isolated from the donor cells and is ready for further manipulation.
The Recombinant Plasmid
To construct the recombinant DNA vector, the genomic DNA must be cleaved to create specific fragments that may be inserted into the plasmid. A DNA library consisting of plasmids containing sequenced fragments of the entire E.coli genome is constructed. It is from this library that the EcoR1 gene may be identified and used to produce vectors that will be inserted into host bacterial cells. A unique restriction enzyme is used to cleave both the plasmid vector and the genomic DNA fragments so that the ends produced are complementary and may be ligated together. In this procedure, it is also important to include alkaline phosphatase to prevent the plasmid from annealing to itself. Alkaline phosphatase removes the phosphate from the 5’ end of the cleaved plasmid. The dephosphorylated end causes directional ligation since the DNA ligase requires the presence of the 5’ phosphate to link the two plasmid ends together. Since the insert still contains the 5’ phosphate it favours the genomic EcoR1 insert to anneal to the linearized plasmid DNA. DNA ligase is used to catalyze the formation of phosphodiester bonds between the 5’-phosphate and the 3’-hydroxy ends of the plasmid and genomic DNA fragments thereby completing the ligation of recombinant plasmid (Old & Primrose 1985). Successful ligation may be determined using agarose gel electrophoresis. The recombinant plasmid migrates a shorter distance on the gel than the smaller, original plasmid, and thus can be identified. The isolated recombinant plasmid is then transformed into competent E. coli cells and is allowed to propagate.
Transformation of Plasmid Vector into Competent Host Cell
The technique administered to transform recombinant plasmids is dictated by the nature of the host cell. E. coli cells are treated with agents that disrupt the cell membrane in order to make the cells competent to take up plasmids (Ninfa & Ballou, 1998). Such agents include calcium chloride that disrupts the cell membrane under high temperatures, causing susceptibility of the cell to transformation. Another method is electroporation, which provides a burst of electric current that disrupts the cell membrane allowing the DNA a brief period to enter the cell before it re-forms (Ninfa & Ballou, 1998).
Selection of Successfully Transformed Bacterial Cells
Successful transformation is identified by plating the bacterial culture on a medium containing an antibiotic. Cells that do not contain the plasmid do not grow in these conditions, as they do not possess the antibiotic resistant gene associated with the plasmid DNA. The type of antibiotic used for the initial selection of successfully transformed cells is dependant upon the nature of the transformed plasmid. For example, pUC19 contains an ampicillin resistant gene. Therefore, ampicillin is used to select these cultures.
A second form of selection identifies cells that contain the recombinant plasmid as opposed to cells containing plasmid with no EcoR1 insert. Again, this selection is dependant upon the nature of the plasmid used; -complementation of pUC19 will be used as a specific example of such selection. In addition to an ampicillin resistance gene, pUC19 contains the amino portion of the Lac Z gene. When this gene is intact, the protein product hybridizes with carboxy portion of the Lac Z gene that is produced by the genomic DNA of DH5 E. coli. The hybridized product is a protein called –galactosidase which hydrolyzes the substrate X-gal thereby producing a blue pigment.
When plasmids contain a DNA insert, the lac Z gene is disrupted and is not transcribed. Thus, functional –galactosidase is not produced and as a result, the X-gal substrate remains uncleaved and no blue pigment is produced. Under these conditions, white colonies are produced. Thus, the -complementation of pUC19 allows the researcher to easily identify colonies containing the recombinant plasmid.
Cultivation of Genetically Modified Bacterial Cells
Batch cultivation is commonly used to grow transformed E. coli for the extraction of the EcoR1 enzyme. In this method, cells are incubated in a closed system with a single batch of medium and allowed to divide to a large quantity. Once the colony has matured and the cells reach a specific optical density of 0.6-0.8 absorbance units, they may be induced (i.e. with IPTG) to express the EcoR1 enzyme. However, if the cells are prematurely induced, the cells may become stressed since metabolic resources required for proper growth are restrained and the plasmid could be degraded. To avoid this degradation, the optical density of the cells is monitored and plasmid gene expression is induced when cells reach the late log growth stage.
A promoter required for the control of EcoR1 expression may be regulated by the presence of a repressor (Sorensen et al., 2005). Transcription of the gene via the promoter may be induced using thermal or chemical inducers (Sorensen et al., 2005). For example, the chemical IPTG binds to the repressor and removes it from the promoter sequence, allowing transcription of the genes. Tamerler et al. (1998) used the chemical isopropyl--D-thiogalactoside (IPTG) to induce production of Eco R1 in E. coli 294 cells. To avoid cell stress caused by the inducer due to excess transcriptional demands, the concentration of the inducer may be slowly increased allowing a gradual build up in the levels of recombinantly expressed protein. Finally, the desired protein may be removed from the host culture using various methods of purification.
Purification Method
In order for the protein to be of any use it must be purified and characterized to ensure it functions properly following recombinant expression.
Since EcoR1 is not excreted, the cell must be disrupted to release the enzyme. Membrane disruption may be completed using a variety of methods including, osmotic disruption, freezing/thawing, digestion of cell walls with enzymes and homogenization. Stronger methods of disruption include high pressure and sonication (Harris et al., 1989). Tamerler et al. (2001) used sonication to disrupt their cell cultures. This method uses sound-wave energy to disrupt and break open the cell membranes. The process of sonication generates heat, so the culture must be kept in a cool bath to prevent protein denaturation (Harris et al., 1989).
After the cells have been disrupted, the extract is cleaned to isolate the desired protein from nucleic acids, and aggregated matter that are also released upon disruption. If these excess compounds are not removed from the extract, they may affect later steps in the purification process (Ninfa & Ballou, 1998). The first step of purification is commonly dialysis, which is used to separate molecules by size. The extract is enclosed in a semipermeable membrane, such as cellophane or nitrocellulose, in the form of a tube that is tied off at the ends making a bag called a dialysis tube (Maniatis et al., 1982). The dialysis tube is placed in a beaker containing dialysate and the pores of the membrane allow small molecules to diffuse outward, such as solvents, salts and substrates while larger molecules, such as proteins are retained inside the structure (Maniatis et al., 1982). At equilibrium, all the small molecules and salts are removed from the dialysis tube, while EcoR1, which is too large to diffuse out of the tube will remain in the bag with other large molecules.
The contents of the dialysis tube are centrifuged to eliminate cell debris and separate the soluble and insoluble components of the sample (Vastola et al., 2000). Centrifugation causes insoluble debris to collect at the base of the centrifuge tube while soluble proteins, like EcoR1 are found in the supernatant and can easily be decanted (Tamerler et al., 2001 & Vastola et al., 2000).
The supernatant is then mixed with a buffer and is applied to column chromatography for further purification. Chromatography separates molecules based on their chemical properties such as molecular mass, charge, and/or solubility (Ninfa & Ballou, 1998). The column is composed of two phases, the stationary and mobile phase (Ninfa & Ballou, 1998). The stationary phase is packed into the column to form a matrix used to separate the molecules in the sample. The mobile phase uses a mixture of the supernatant and a buffer to move the supernatant through the column. Interactions between the matrix and proteins in the supernatant retain the proteins in the column. By changing the concentration or composition of the buffer, the proteins are released from the column in a number of fractionations (Ninfa & Ballou, 1998). The unique properties of each protein allows the fractions produced to consist almost entirely of a specific protein. The fractions with the highest enzyme concentration may then be pooled.
Tamerler et al., (2001) used ion-exchange chromatography to isolate EcoR1 from the supernatant mixture. This method applies phosphocellulose and hydroxyapatite as the stationary phase in the column, and a mixture of supernatant and buffer of increasing NaCl as the mobile phase. Ion-exchange chromatography separates molecules based on their charge groups. Molecules in the supernatant that carry a charge opposite to that of the matrix interact electrostatically with the stationary-phase medium (Scopes et al., 1987). As the buffer moves through the column in concentrations of increasing counterion, it displaces molecules attached to the matrix. Molecules with smaller charges are weakly attached to the matrix and are therefore released first in the presence of the buffer containing small concentrations of counterion (Ninfa & Ballou, 1998). As the concentration of counterion increases, molecules with larger charges that strongly interact with the matrix are consecutively displaced. The fraction containing the protein of interest may be determined from other fractions based on the chemical properties of the protein. This method is used only to purify ionized molecules since charged molecules will bind to the stationary phase and their movement will be retarded within the column (Ninfa & Ballou, 1998).
Hydroxyapatite column chromatography is similar to the phosphocellulase chromatography with the exception that the buffer contains increasing concentrations of phosphate. Each fraction is then tested to determine if the desired protein is present. Once the fractions containing the target protein are found, the protein may be further characterized.
To test the homogeneity of the purified protein, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is performed. Electrophoresis separates molecules based on their charge and size (Ninfa & Ballou, 1998). The distance of protein migration through the gel matrix is determined by the charge to mass ratio of the molecule where large neutral molecules move slowly and small charged move quickly (Vergnon et al., 1999). Proteins may pose a problem for this type of separation since proteins of different size and charge may migrate at the same rate (Ninfa & Ballou, 1998). To overcome this problem, the proteins are denatured with a detergent, such as SDS, which solubilizes hydrophobic portions of the protein and gives the molecule an overall negative charge. This allots the same charge to mass ratio to all proteins and allowing them to be separated only by size (Vergnon et al., 1999). Figure 3 is an example of an SDS-PAGE gel, each lane is a fraction from column chromatography. It can be seen that with each fraction the band representing the desired protein gets larger. As the concentration of the desired protein increases in each fraction, the number of undesired proteins decreases. This is an indication that the desired protein is undergoing purification (Ninfa & Ballou, 1998). To be certain the band of increasing concentration is the desired protein, a commercially produced version is purchased and is run on the gel as a control (Vergnon et al., 1999). In Figure 3 the commercially purchased protein is on the right hand side and produces a dark band approximately half-way down the gel. As the band of increasing concentration (increasing thickness and darkness) are also situated approximately half-way down the gel, it may be concluded that the desired protein is contained in these bands.


Figure 3. Example of SDS-PAGE. The gel illustrates proteins contained in fractions collected over time from column chromatography. One protein band becomes larger in size with each fraction and when compared to the commercially bought protein (far right well), the indication is that the target protein is contained in these bands (Ninfa & Ballou, 1998).
Following the separation of EcoR1 by SDS-PAGE gel electrophoresis, an assay is performed to measure the activity and amount of protein extracted from the host cell. Activity may be tested using enzyme kinetics, which provides information about how the enzyme responds to substrates (Ninfa & Ballou, 1998). Michealis-Menton anaylsis can determine the kinetic parameters of an enzyme (Vmax and Km). For the Vmax of EcoR1, this process consists of taking a constant amount of enzyme and exposing it to different amounts of DNA to determine how long it takes the enzyme to cleave the DNA. Eventually the enzyme reaches Vmax and is saturated by the concentration of DNA and will no longer increase its rate of conversion of substrate to product. The Michealis Menton constant (Km) is the substrate concentration at which the reaction rate is half-maximal and this parameter is an indicator of the affinity of the enzyme for its substrate.
The amount of extracted protein is quantified by colourimetric methods. In Bradford and Pierce assays, the protein binds to the dye reagent and changes the spectrophotometric properties of the dye. This enables quantification when compared to a standard curve of known proteins (usually Bovine Serum Albumin, BSA). By using these methods of purification and characterization Tamerler et al. (2001) found that the recombinant strain of E. coli 294 was an optimal source of EcoR1 producing 3.43 x 106 U/ g-wet cells.
The quality of the enzyme is then tested to ensure the enzyme is functional and suitable for commercial production. Tamerler et al. (2001) used an overdigestion assay to determine the purity of the sample by testing for activity of endonucleases or exonucleases that may have contaminated the sample. This is completed by comparing the digestion patterns of varying concentrations of EcoR1 over varying periods of time. If the sample is pure, the patterns of digestion are identical for each concentration of enzyme over each period of digestion. However, if the sample is contaminated, similar patterns of digestion will not occur.
The cut-ligate-recut method was also employed by Tamerler et al. (2001) to determine if the purified EcoR1 cleaved DNA in the fashion attributed to the isolated enzyme. To do this, Tamerler et al. (2001) used the isolated EcoR1 to cleave DNA. The cleaved fragments were then ligated using a T4 ligase. The ligated fragments were then recut using the EcoR1 enzyme employed in the first cleavage. The digested DNA from the first and second cleavages were run on an agarose gel and compared to determine whether the enzyme properly digested the DNA. If the band patterns produced by each cleavage were the same, it was concluded that the enzyme was cutting in the same place for each cleavage event and was therefore working properly.
Once the EcoR1 has been successfully produced, purified and characterized, it is then stored for use. Proteins are usually stored at low temperatures such as –80oC to prevent denaturation (Ninfa & Ballou, 1998). Cryogenic agents, such as salts and glycerol, may be added to stabilize the protein during freezing and thawing during laboratory use (Ninfa & Ballou, 1998). Following the successful production, purification, characterization and storage of the enzyme, it may be shipped throughout the world for research purposes.

Literature Cited:

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Water, P. (2002). Molecular Biology of the Cell. 4th Ed. Garland Science. Pp. 493.

Danna, K., and Nathans, D. (1971). Specific cleavage of simian virus 40 DNA by Restriction Endonuclease of Hemophilus Influenzae. Proc. Nat. Acad. Sci. USA. Vol. 68, No. 12, pp. 2913-2917.

Harris, E.L.V. and Angal, S. (1989). Protein purification methods: a practical approach. IRL. Press, Oxford

Lederberg, S. (1965). Genetics of Host-Controlled Restriction and Modification of Deoxyribonucleic Acid in Escherichia coli. J. Bacteriol. Vol. 91, No. 3, pp. 1029-1036.

Linn, S., and Arber, W. (1968). Host Specificity of DNA produced by Escherichia Coli, X In Vitro restriction of phage fd replicative form. Biochemistry. Vol. 59. pp. 1300-1306.

Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982). Molecular cloning. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

Ninfa, A.J., and Ballou, D.P. (1998). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Fitzgerald Science Press, Inc. Bethesda, Maryland.

Old, R.W., and Primrose, S.B. (1985). Principles of gene manipulation: an introduction to genetic engineering. Blackwell Scientific Publications. Oxford, Boston.

Pingoud, A., and Jeltsch, A. (2001). Structure and function of type II restriction endonucleases. Nucleic Acids Research. Vol. 29, NO. 18. pp. 3705-3727.

Scopes, R.K. (1987) Protein Purification. Principles and Practice, 2nd ed. Springer-Verlag, New York

Sorensen, H.P. & Mortensen, K.K. (2005). Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology. 115: 113-128

Tamerler, C., Onsan, Z.I. & Kirdar, B. (2001). A comparative study on the recovery of EcoR1 endonuclease from two different genetically modified strains of Escherichia coli. Turkish Journal of Chemistry. 25:63-71

Vastola, M. (2000). Centrifuges: How they operate and how to select one. Filtration and Separation. 37:16–19

Vergnon, A.L., and Chu. Y.H. (1999). Electrophoretic methods for studying protein-protein interactions. Methods. 2: 270-277

Vogel, T., Gluzman, Y., Kohn, N. Altered restriction endonuclease cleavage pattern of Simian virus 40 DNA. J. Virol. Vol. 29, No. 1, pp. 153-160.