Protein purification is a series of processes to isolate a protein from a complex mixture. It is important to characterize a protein's function, structure, and interactions. The general steps of purification include preparing the source, exploiting differences in protein properties through separation techniques, and monitoring purity with assays. Purification yields a pure protein sample for determining its sequence, structure, and role. Affinity tags and other tags are often used to aid purification. Biopharmaceuticals are medically important proteins produced through biotechnology.

1. PROTEIN
PURIFICATION
By Inder Ishwarlal Gohri

2. What is Purification ?
01.
Why their is a Need of
Purification ?
02.
.
03. General purification steps.
04. Preparation of Source.
05. Biopharmaceuticals
06. Protein Tags
07. Quantification of Proteins
(Assay Methods)
08. Monitoring the activity of
purification
Topics

3. What is Protein Purification?
 Protein purification is a series of processes intended to isolate one or a
few protein from a complex mixture usually cell ,tissues or whole
organisms.
 Protein purification is vital for the characterization of the function, structure
and interactions of the protein of interest.
 Purification should yield a sample of protein containing only a particular
type of molecule, the protein in which the biochemist is interested.
 Separation steps usually exploit differences in protein size, physico-
chemical properties, binding affinity and biological activity.
 The pure result may be termed protein isolate.

4. Why there is need of protein
purification ?
From pure protein,
a)We can determine amino acid
sequences.
b)Evolutionary relationship between
proteins in diverse organisms.
c)And we can investigate a protein’s
biochemical function.

5.  Also crystals of the protein may be grown from pure
protein, and from such crystals we can obtain x-ray data
that will provide us the protein’s tertiary structure and the
actual functional unit.
Figure 2. Workflow for protein structure determination using X-ray crystallography.

7. a)
Preparation
of the source
b)
Knowledge
of protein
properties
c)
Development
of an Assay
d) Primary
Isolation
e) Final
Purification

8. Preparative purifications aim to
produce a relatively large quantity
of purified proteins for subsequent
use.
• Examples :such as enzymes (e.g.
lactase), nutritional proteins (e.g.
soy protein isolate), and certain
biopharmaceuticals (e.g.insulin).
Analytical purification -small
amount of a protein for a variety of
research or analytical purposes,
including identification,
quantification, and studies of the
protein's
•structure, post-translational
modifications and function.
•Examples : Pepsin and urease

9. Biopharmaceuticals
Biopharmaceuticals are medical drugs – proteins,
antibodies and nucleic acids that are produced using
biotechnology and are used for therapeutic or in vivo
diagnostic purposes.
The first biopharmaceutical agent was insulin that was
approved for human use in 1982.
Today, over 160 biopharmaceutical agents are approved
in the USA

10. Protein tags
Affinity tags
Affinity tags are appended to proteins so that they can be purified from their crude
biological source using an affinity technique. They act as solubilization agent
Ex:
1.chitin binding protein (CBP),
2. maltose binding protein (MBP),
3. glutathione-S-transferase (GST).
4. The poly(His) tag is a widely-used protein tag
Solubilization tags
Solubilization tags are used, especially for recombinant proteins expressed in E. coli, to
assist in the proper folding in proteins and keep them from precipitating.
Ex: These include thioredoxin (TRX) and poly(NANP).

11. Chromatography tags
Chromatography tags are used to alter chromatographic properties of the protein to afford
different resolution across a particular separation technique. Often, these consist of
polyanionic amino acids.
Epitope tags
Epitope tags are short peptide sequences which are chosen because high-affinity antibodies
can be reliably produced in many different species. These are usually derived from viral
genes, which explain their high immunoreactivity
Ex.Includes V5-tag, c-myc-tag, and HA-tag.
Fluorescence tags
Fluorescence tags are used to give visual readout on a protein.
Ex.GFP and its variants are the most commonly used fluorescence tags.

12. Quantification of Proteins
• A method of determining in a quantitative fashion the amount of a particular
activity present is
Assay methods:
• If the protein is an enzyme, the assay of choice measures that protein‘s ability to
convert a substrate to a product, where, for signal-to-noise reasons, appearance
of product is preferred over disappearance of substrate.
• When the protein of interest lacks enzymatic activity, that protein may be quantitated
immunologically
SDS polyacrylamide
Western blot

13. • The ratio (specific activity) of enzyme activity units (or
chromophore absorption value) divided by a measure of total
protein content is then used to estimate relative purity at all stages
of purification.
• Copper-based Protein Assay Chemistries
1. Biuret Reaction
2. Bicinchoninic Acid (BCA) Protein Assays
3. Lowry Protein Assays
• Dye-based Protein Assay Chemistries
1. Coomassie Dye (Bradford) Protein Assays
2. Pierce 660nm Protein Assay
Colorimetric assays

14. • The Bradford assay involves the binding of the Coomassie
Brilliant Blue G-250 dye to protein.
• The dye binds more favourably to basic residues like arginine
Protein assay by Bradford Assay

15. Total protein mg/mL
• Quantity of protein present in the fraction
Total activity (units of activity)
• Use a portion of sample to determine activity.
• Multiply activity by total volume to determine
total activity.

16. • Specific activity (units of activity/mg)
S.A = Total activity of E
Total protein
• % yield: measure of activity retained after each step in procedure.
Percentage yield = Total activity at particular step
Total activity of initial extract
• Purification level increases at each step of purification
Purification = S.A at particular step
S.A of initial crude extract

17. REFERENCE :
1.https://www.slideshare.net/PradeepNarwat/purifi
cation-of-proteins-purification-of-enzymes
2.https://youtu.be/PVvpEKeOzEM