Proteins have many important functions in living organisms. The document discusses protein extraction and purification methods. It describes how proteins are first extracted from tissues or cells using mechanical or chemical techniques like homogenization and centrifugation. Several common purification techniques are also explained, including salting out, dialysis, differential centrifugation, and column chromatography. The goal of purification is to isolate a single protein from a complex mixture to study its structure, function, and interactions.

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UNIVERSITY OF JUBA
SCHOOL OF APPLIED & INDUSTRIAL SCIENCES
P.O. 82, JUBA-SOUTH SUDAN
SECOND YEAR SEMESTER THREE
2020/2021 ACADEMIC YEAR
GENERAL BIOCHEMISTRY PRESENTATION WORK
PREPARED BY GROUP B
UNDER THE INSTRUCTION OF MR. BOJO OPENZI
SATURDAY, FEBRUARY 6TH 2021
PRESENTATION QUESTION

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“Explain the Functions, Extraction and Purification of Proteins”
1. Greetings and Invitation
With the great honor and humility, the entire membership is so delighted at this long
awaited day to convey its best wishes for the new month to Instructor, Mr. Bojo
Openzi and the class as a whole not forgetting success wishes to all colleagues in the
forthcoming semester III examinations.
At the meantime, the group cordially invites the attention and company of the entire
class as it runs through the aforementioned question in the next few minutes.
2. Introduction to Proteins
Before we get to know all about the above stated question, let’s first know what a
protein is. The word protein comes from a Greek word called “proteios” meaning
“primary” or “holding the first place”.
Therefore, protein is a natural occurring organic substancewith extremely complex
structural organization, which is made up of amino acids joined in a chain by the aid
of peptide bonds.
They are the primary substituents of molecules of all the living organisms and these
includes the enzymes, hormones and the antibodies.
It was discovered and named by a Swedish Chemist called “Jons JacobBerzelius”
in 1838 and then later same year, a Dutch chemist called “GerardJohann Mulder”
found out that proteins are made of amino acids.
3. Functions of Proteins
Not limited to the following, the functions of proteins include:
 Stimulation of biochemical reactions

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Proteins in the form of enzymes chiefly catalyzes the rate of biochemical reaction in
the body of any living organism. They performs their functions repeatedly working as
the “the workhorses” that carry out the processes oflife.
E.g Maltose + Water Maltase Glucose + Fructose
 Provision of structural support
Proteins such as the keratin, collagen, fibrin and many other elastic proteins help
provide structural supportto animals, insects and spiders respectively.
 Provision of Energy
Proteins serve as the sources of energy to all those organisms that don’tmake their
own food. Furthermore, they as well provide energy to large living organisms
especially the animals during the scarcity of carbohydrates in their bodies.
 Growth and Maintenance of Tissues
Proteins are necessarily required for the growth and maintenance of tissues of any
living organisms.
 Bolstering of the body’s immune system
Proteins form antibodies which provide protection to mainly the animals’ bodies from
foreign invaders suchas the disease-causing bacteria and viruses.
 Maintenance of proper pH level
Proteins act as a buffer system, helping the living organisms maintain their properpH
values alongside other bodily fluids.
 Medium of Transport
Proteins provide transportation of substances from one part of an organism to another.
For instance; Haemoglobin, an iron-containing protein in vertebrate blood helps
transport oxygen from the lungs to other parts of the body. Other proteins do carry
molecules across cell membranes.
 Provision of cellular communication

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Proteins also play a very crucial role in appropriate communication between the cells
of an organism. For example, protein receptors built into the membrane of the nerve
cell detect signaling molecules released by the nerve cells hence making the cell
responds to chemical stimuli.
 Regulation of glucose level
Hormonal protein such as insulin, stimulate other tissues take up glucose if a given
tissue contain high level of glucose hence regulating blood sugar level.
 Balancing of fluids level in the blood
Protein regulate bodyprocesses to maintain the fluid balance in the bodyof a given
living organism. For example, proteins such as albumen and globulin in our blood
help maintain our body’s fluids balance by attracting and retaining water.
 Reduction of appetite and hunger levels
Studies have shown that protein reduce the level of the hunger hormone ghrelin
hence decreasing the level of hunger and indeed, the level of appetite for food.
Remember!!!
Proteins speak,
“We are the bases for the structure and function of life, composedoftwenty amino
acids, the building blocks; organized into primary, secondary, tertiary and quaternary
structures and classified as simple, conjugated and derived proteins”.
4. Extraction of Proteins
This is the process ofobtaining proteins from the various sources such as the plant
tissues and the animal tissues or any other available source.
It is extracted from the said sources for the purposeof study mainly in research
laboratories and as well as for industrial use for the productionof energy-giving
foodstuffs.
Proteins initiate and mediate a thousands of biochemical pathways that govern an
organism’s bodyfunction, thus there is a need for a careful study about them. This is

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because if we do so, we’ll get to understand about how our bodies function, how the
disease enters into the bodyas well as the expression of the genetic code.
The major challenge here to overcome when studying proteins is the choice for the
most appropriate method of protein extraction.
Protein extraction mainly starts with mechanicalor chemicaltechniques. The initial
steps of protein extraction often involve crude mechanicaldisruption such as
cutting, smashing or shearing tissue into smaller pieces. If intracellular proteins are
the target, then detergents can be used to help break apart the phospholipid cellular
membrane.
A. Protocols forprotein extraction
Extraction is done from tissue or cell culture. Tissue requires more steps as there are
more layers and heterogeneity across the sample. The following steps are crucially
taken when isolating or extracting proteins from the various sources selected.
A. Mechanicalhomogenizationofthe given selectedsample
For tissue, the first step is to mechanically homogenize the sample. Next, the
centrifugation will give you cytoplasmic proteins. Further, you can use chemical
buffers to get nuclear proteins. Another chemical buffer containing detergents can be
used to obtain membrane proteins.
For cell culture, one needn’t to mechanically-homogenize his/her sample but the rest
of the procedureremains the same, and he/she begins with lysing his/her cells. All
buffers used here, may need to be optimized for the different situations and it is vital
to consider the presence of proteases in our sample. Kits that include buffers for
protein extraction are usually available commercially or can be prepared in
laboratories which routinely do protein extraction.
B. Centrifugation
Large pieces of tissue are easily removed by centrifugation after the crude initial
tissue disruption. Proteins may therefore be extracted from particular cell
compartments via multiple rounds of centrifugation. Additionally, nucleic acids can
be chemically precipitated out of the initial tissue slurry and removed by
centrifugation. A similar technique can be applied to precipitate proteins out of the
supernatant. Protein interaction and function are highly dependent on three

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dimensional structures. Thoseamino acid side chains provide the chemical pockets
that mediate that function via hydrophilic and hydrophobic interactions including
hydrogen bonding, ionic and Vander Waals forces.
B. Affinity
Amino acid side chain can be exploited to aid protein extraction. An example is an
ion-exchange column or a hydrophobic column. There are amino acids with
positively charged side chains (arginine, histidine and hysine), negatively charged
side chains (aspartic acid, ghitamic acid) and hydrophobic side chain (tyrosine,
tryptophan, phenylalanine, valine, isoleucineete). These affinities allow proteins to
adsorb to a column consisting of a positive, negative or hydrophobic matrix. Once
adsorbed to the column, the proteins can be washed and eluted as an enriched sample.
C. Other Methods
The recent methods of protein extraction i.e centrifugation and affinity columns are
believed to be the most effective for the general isolation of proteins in bulk.
However, if the experimental goal involves the extraction of a specific protein, then
there are other methods that maybe more appropriate. This is particularly helpful for
antibody or antigen extraction as these proteins have specific affinities for each other.
Alternatively, specific antibodies can be isolated by attaching their corresponding
antigen to a solid surface such as a column or bead. Similarly, the extraction of a
specific antigen can be completed by using antibodies containing a recognition site or
that antigen. This process is made quick, easy and efficient with the use of
functionalized super paramagnetic nanoparticles and bio-magnetic separation.
PROTEIN PURIFICATION
Protein Purificationis a series of process intended to isolate oneor a few from a
complex mixture, usually the cells, tissues or the whole organism.
WHY DO WE PURIFY PROTEINS
 To identify the structure and function of the proteins of interest.
 To obtain a pure protein that can be of an industrial interest, i.e used in
production of energy-giving foodstuffs as well as the vaccines.
 To know their composition as well as their interactions with each other.

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The purification process helps separate the protein and non-protein partsof the
mixture from each other, and finally separate the desired protein from all other
proteins. Separation of one protein from the rest is typically the most laboriousaspect
of protein purification.
The separation steps are usually determinable through the differences in protein size,
physical & chemical properties, binding affinityand biological activity. The pure
result is then termed protein isolate.
TYPES OF PROTEIN PURIFICATION METHODS
1. Saltingout
Most proteins are less soluble at high salt concentrations, an effect called salting. The
salt concentrate at which a protein precipitates differs from one protein to another.
Hence salting out can be used to fractionate proteins. For example, 0.8M ammonium
sulfate precipitate fibrinogen a blood clothing protein, whereas a concentration of
2.4M is needed to precipitate serum albumin.
Salting out is also useful for concentrating dilute solutions of protein, including active
fractions obtained from other purification steps.
Dialysis

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This is the method of separating molecules of differing sizes by differential diffusion
through a semi-permeable membrane.
Proteins can be separated from small molecules by this. This technique is as well
useful in removing the salt or other small molecules from their mixtures.
The diagrambelowshows how this process of dialysiscanbe used to purify
proteins.
2. Differential Centrifugation
Here, an electronic device called a “Centrifuge” is use to separate proteins (or any
other material) on the basis of their sizes, masses, and densities.
3. Column chromatography
This is the best approachsuitable for the purification of larger amounts of proteins,
for it perfectly filters out the desired proteins at the highest accuracy.
The method requires no much efforts in the setup and in carrying out the whole job.
It’s the best and the standard method used in pharmaceutical industry for the
production of the protein-containing chemicals such as the vaccines, among others.
Precipitation and differential solubilization
Ammonium sulfate precipitation.
In bulk protein purification, a common first step to isolate proteins is precipitation with
ammonium sulphate (NH4)2 SO4. This is performed by adding increasing amount of
ammonium sulfate and collecting the different fractions of precipitated protein.
Subsequently, ammonia sulphate can be removed using dialysis. During the ammonium
sulphate precipitation step hydrophobic groups present on the proteins are exposed to
the atmosphere, attracting other hydrophobic groups; the result is form actionof an
aggregate of hydrophobic components. In this case, the protein precipitated will

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typically be visible to the naked eye. One advantage of this method is that it can be
performed in expensively, even with very large volume.
 Ultra centrifugation;
Centrifugation is a process that uses centrifugal force to separate mixtures of particles
of varying masses ordensities suspended in liquid. When a vessel containing a mixture
of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds,
the inertia of each particle yields a force in the direction of the particles velocity that is
proportional to its mass. The tendency of a given particle to move through the liquid
because of this force is offset by the resistance the liquid exerts on the particle. The net
effect of “spinning” the sample in a centrifuge is that massive, small, and dense
particles move outward faster than less massive particles or particles with more “drag”
in the liquid. When suspensions of particles are “spun” in a centrifuge, a “pellet” may
form at the bottom of the vessel that is enriched forthe most massive particles with
low drag in the liquid.