EXTRACTION, PURIFICATION AND ASSAY OF INVERTASE AND INVERTASE INHIBITOR FROM CEREAL GRAINS

 

5 Protein isolation assignment

Due Date:               May 18th

Weight:                  20%

Length:                   Approx 2000 words please include a word count

Each student will be assigned a different protein for this assignment at the intensive school. You will research your protein to find out:

  • Where it occurs; what organism(s), organs, cell type, organelle?
  • Its biological function in the context of the whole organism.
  • Properties of your protein which might be useful for isolation and assay

You will use the information you have found to plan an extraction, purification and assay protocol.

At the intensive school, you will be given detailed instruction on the use of journal and protein databases to find relevant information as well as explanation and instruction on how to use this information to isolate your protein.

You should also look carefully at the practical manual; the section on “EXTRACTION, PURIFICATION AND ASSAY OF INVERTASE AND INVERTASE INHIBITOR FROM CEREAL GRAINS” is written partly as a model for this assignment.

You are encouraged to discuss your protocol with staff prior to submission of your assignment.

Data sheet (to be completed and submitted at the intensive school).

NOTE a Word version of this table is available separately. You should copy this and fill in the relevant details.

Please insert URLs for UniProt accession and EC number as hyperlinks.

Name of protein.

Be specific e.g. bovine hexokinase 1 NOT hexokinase

Citation

UniProt accession

If your protein has more than one different subunit there will be a different code for each subunit.

URL

EC number

Only relevant if your protein is an enzyme

URL

Protein function

Be brief and specific e.g. an equation for reaction catalysed

e.g. glucose + ATP -> glucose-6-P

Citation

Physiological process

Give a brief summary of the pathway or process in which your protein is involved (must be in your own words):

E.g. glycolysis, glucose signalling

Citation

Location

Include all relevant from: organism, organ, cell type, subcellular location or extracellular fluid

Citation

Membrane protein

Is your protein an integral membrane component or membrane associated?

Citation

MWt

Include molecular weight of full protein not one subunit.

Citation

pI

Include experimental pI of entire protein if possible. Use predicted pI only if expt is not available

Citation

Subunits

Include number and type of subunits

Citation

Post-translational modification

Include any post-translational cleavage, glycosylation, phosphorylation known or predicted  (instructions provided at intensive school)

Citation

Ligand(s)

Include any molecules your protein can bind to

Citation

Cofactors

List any required cofactors

Citation

pH Optimum

check http://www.brenda-enzymes.org/

Citation

Ammonium sulfate fraction

check http://www.brenda-enzymes.org/

Citation

Thermal stability

check http://www.brenda-enzymes.org/

Citation

Factors which enhance stability

check http://www.brenda-enzymes.org/

Citation

Assay

How could your protein be measured (be brief e.g. enzyme assay; state substrate or product measured and how (e.g. wavelength) OR ELISA etc.

Citation

Include a full list of references. 

Except for UniProt accession and EC number these will be journal references NOT database URLs.

 

Assignment report

The assignment report will be divided into three main sections which should appear clearly in your report:

  1. Introduction: (Approx 750 words)

This section sets out background information about the protein you have been given. This should include:

A description of where your protein is found:

Many proteins are found in a large number of different organisms and sometimes there are several different forms of the protein in different parts of the same organism. Before writing your report you must decide which organism/tissue/organelle you are going to use for your preparation and focus the bulk of your introduction to describing the function of your protein in that particular organ/organism. In addition, information on the tissue/organelle location of your protein is essential for its efficient isolation eg you may wish to start your method by making a heart muscle mitochondrial preparation.

The function or role of your protein in the organism:

Please make sure you put enough detail in this section. You should describe not just the immediate function of your protein (eg. the reaction it catalyses), but how this is important to the function of the cell/organism. For example you need to discuss the pathway in which your enzyme participates, or the role of your hormone and the metabolic pathways it affects etc. Note: a knowledge of the function of your protein may provide you with the information you need to assay it.

 

  1. Purification protocol  (approx. 1000 words)

This will be the most comprehensive component of the report and will include several sections. Look at your practical manual (isolation procedure) for a model of how to structure this section, amount of detail and the type of information required.

Starting material and cell lysis

First you must extract your protein. Start with your choice of organism/tissue. Describe how you are going to break open the cells to remove your protein. You also need an extraction solution. Did you discover anything that increased/decreased your enzyme stability that should be included/excluded. What pH was your enzyme stable at etc. If you have a extracellular protein (e.g. blood plasma) you don’t have to extract it but you do have to remove the cellular fraction. (NOTE you do not have to provide concentrations or volumes of extraction buffer)

Organelle isolation and/or
removal of cell debris

Once you have homogenised/lysed your tissue you will need to include at least one centrifugation step to remove unwanted cell debris. When describing centrifugation you should clearly indicate whether the fraction you want is in the pellet or the supernatant. Your extraction section will often include the preparation of an organelle or membrane fraction. There is no shortage of references detailing how to prepare mitochondria, chloroplasts, ER etc. You may also need to consider whether you need to lyse your organelle. Finally if you have an insoluble protein e.g. (membrane protein), look out for information on how can you solubilise it.

Protein purification

At this stage you have a cell-free extract containing your protein as well as many others. We now want you to describe 3 to 4 purification steps used to separate your protein from others in the crude extract. These should use a variety of different properties of your protein to separate it e.g. you may want to use solubility, then size, then charge to isolate your protein. You should describe each step in reasonable detail; e.g. provide the concentrations of ammonium sulphate used, the grade of gel filtration material, the pH at which ion exchange was carried out, the ligand used for affinity chromatography. (NOTE concs and volumes are NOT required). You are encouraged to use spin columns chosen from the information in the appendix to this document.

You MUST explain the rationale for the choice of each step i.e. what properties of your protein are you using as the basis for a particular step. Look at practical manual for guidance.

 

  1. Assay (approx 250 words )

Assay

You must have a way of measuring your particular protein. Note this is different from measuring total protein via e.g. Bradford assay. The assay will relate to particular biological or physical properties of your protein. If it is an enzyme your assay will normally be a measurement of its catalytic activity. Other common assays are immunological assays (eg. radioimmunoassay or an ELISA) or a bioassay (for a hormone).

Note: you need to explain the assay and describe how it is relevant to your protein.

  1. References (marks included in relevant section)

Sources of information must be correctly cited in the appropriate places in your report. EACH piece of information in ALL sections of your assignment MUST be referenced. References must be cited in the text as well as listed in alphabetical order by author in a reference list.

You should always cite primary references; this means the journal articles that reported the original finding. Other internet sources, the practical manual are not acceptable references.

Students are strongly encouraged to use Endnote Web for referencing. This will be demonstrated at the intensive school.

How to obtain information:

At the intensive school you will be shown how to search for specific information on your protein from journal databases and protein databases.

Web of Science – available via the library web page

Google scholar – available via the library web page

BRENDA http://www.brenda-enzymes.org/

Uniprot http://www.uniprot.org/

The Protein Data Bank http://www.rcsb.org/pdb/home/home.do

More general information can be found in the following books:

   -Methods in Enzymology

   – Biochemistry, Voet & Voet, 2004 p127-154

   -Experiments in Biochemistry, A hands on Approach, Farrell, SO & Taylor LE, 2006.

   -Modern Experimental Biochemistry, Boyer R.F. (1986)

–  -Protein purification protocols, Ed. S. Doonan (1996).

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