CHEM12: MIDTERM

Week 7 PROTEIN PURIFICATION AND CHARACTERIZATION TECHNIQUES & Week 8 ENZYMES , Week 9: CARBOHYDRATES

Proteins

are polymers composed of hundreds or even thousands of amino acids linked by peptide bonds

PROTEINS THREE BASIC CLASSES

1.  Fibrous Protein

2. Globular Protein

3. Membrane Proteins

Proteins fall into three basic classes according to shape and solubility:Fibrous Protein

Collagen

Proteins fall into three basic classes according to shape and solubility: Globular Protein

Myoglobin

Proteins fall into three basic classes according to shape and solubility:Membrane Proteins

Bacteriorhodopsin

Fibrous Protein

• Play structural roles in the cell (matrix formation)

• Often water Insoluble

Fibrous Protein

Regular linear structures

Globular Protein

• Roughly spherical in shape

• Often water soluble

 Membrane Protein

• Often water insoluble but soluble in detergent

 Membrane Protein

• Hydrophobic amino acid side chains present outside

• Play role in the cellular transport

Globular Protein

• Hydrophobic amino acid side chains present in the interior of the molecule while hydrophilic side chains are outside exposed to the Solution

ORGANIZATION OF PROTEINS

Primary Structure

Secondary Structure

Tertiary Structure
Quaternary Structure

Primary Structure

Stabilized by peptide bonds only

Secondary Structure

-α–helix, β-sheet, β-bends, motifs etc.

Secondary Structure

-Stabilized by covalent (peptide, disulfide) & non-covalent interactions (H-bonding, electrostatic forces, Hydrophobic interactions and Van der wall forces).

Tertiary Structure

-Stabilized by covalent & non-covalent interactions

Tertiary Structure

-More complex form of primary structure

Quaternary Structure

Consist of two or more polypeptides linked to each other by covalent and non-covalent interactions.

Purity

is defined by the general level of protein contaminants and also by the absence of contaminants of special interests such as microbes, toxins etc.

Protein purification five stages:

1. Preparation of sources

2. Knowledge of protein properties

3. Development of an assay

4. Primary isolation

5. Final purification

WHERE DO WE GET PROTEINS?

•Whole organisms

• Organs or tissues

•Embryos

•Tissue culture cells

•Proteins from expression systems

PREPARATION OF SOURCES:

The amount of protein can be increased by increasing cultivation volume.

PREPARATION OF SOURCES:

The raw materials from which proteins can be isolated such as microbial culture or animals or plant sources should be selected.

KNOWLEDGE OF PROTEIN PROPERTIES

one should know about different properties of proteins such as-

• intracellular 

• extracellular occurrence, 

• denaturation temperature, 

• pH range

• ionic stability,

• molecular weight, 

• Charge,

• iso-electric point, 

• binding partners

KNOWLEDGE OF PROTEIN PROPERTIES:

-Before employing any procedure

DEVELOPMENT OF AN ASSAY:

-An assay developed should be 

• convenient

• easy

• rapid

• precise for purification.

SEPARATION STRATEGIES

-Before a protein can be analyzed, it must first be isolated in a pure state, i.e., purified.

Protein purification

- means separating the protein of interest from other proteins and components

Protein purification

• Extraction

• Precipitation

• Purification

• Concentration

• Storage

Protein can originate from different sources like:

• Physiological 

• liquids, 

• Tissue,

• cell lines,

• microbes in which protein usually resides or is expressed after genetic manipulation.

Protein Isolation: Methods for isolation

1. Cryogenic Grinding (Grinding in liquid nitrogen)

2. Ultrasound homogenization

3. French Press

4. Lysis buffer

Cryogenic Grinding (Grinding in liquid nitrogen)

-Used for hard tissues and cells like roots, and stems, but also for hard-walled cells like some algae and cyanobacteria.

Cryogenic Grinding (Grinding in liquid nitrogen)

-Low temperature protects the proteins during grinding

-Time-consuming (manual grinding) or requiring suitable machinery.

Ultrasound homogenization

-Used for soft tissues like some leaves, and a post-treatment after grinding

Ultrasound homogenization

-Does not require freezing and thus may avoid artifacts of freezing, but may cause artifacts by heating of the sample.

Cryogenic Grinding (Grinding in liquid nitrogen)

Ultrasound homogenization

Fibrous Protein

 Globular Protein

Membrane Protein

French Press

-Used for individual cells (plant cell culture, algae or bacteria) without or with soft walls.

-Does not require freezing and thus may avoid artifacts of freezing.

French Press

-Requires very expensive machinery.

French Press

Lysis buffer

Lysis buffer

-Used only for bacteria or animal cells

-May cause degradation

Lysis buffer

-No machinery needed.

PRIMARY ISOLATION:

This consists of separation of protein from other cellular components and done in different methods:

PRIMARY ISOLATION: different methods:

-Concentration

-Cell lysis: (For intracellular protein)

-Refolding

Cell lysis: (For intracellular protein)different methods

-Physical method

-Chemical method

-Enzymatic method

Ultrafiltration

is a membrane filtration in which hydrostatic pressure is applied which

causes movement of solution across the semi-permeable membrane.

Ultrafiltration

are usually used to concentrate extracellular proteins from cell.

Physical method:

-Mechanical method: Bead mill, Homogenizer, Microfluidizer, Sonicator, French press/ X-mesh

-Non-mechanical method: Decompression, Osmotic shock, Thermolysis, Freeze thaw, Dessication, Cell bomb

Chemical method:

-Chemical permeabilizer whents

-Antibiotics

-Detergents

-Chartrops

-Chelating agents

-Hydroxides and hypochlorides

Enzymatic method:

-Autolysis

-Lytic enzyme

-Phage mediated lysis

PROTEIN PURIFICATION : AN OVERVIEW OF PRINCIPLES

-Separation by expression site

-Separation by size

-Separation by charge

-Separation by specific binding sites

Separation by expression site

-Selective use of tissues or organelles

-Separation of soluble from membrane proteins by centrifugation

Separation by size

-Ultrafiltration

-Size exclusion chromatography

-Preparative native gel electrophoresis

Separation by charge

-exchange chromatography

-Isoelectric focusing (as chromatography, in solution or in gel electrophoresis)

Separation by specific binding sites

-Metal affinity chromatography using natural or artificial metal binding sites

-Immuno-Chromatography using immobilized antibodies

-Magnetic separation using magnetically tagged antibodies

CHROMATOGRAPHY

It is a method to separate molecules according to their distribution between the mobile and stationary phases.

CHROMATOGRAPHY

is the usual method for obtaining pure protein.

Different types of chromatographic methods:

-size exclusion chromatography (SEC)

-Ion-exchange chromatography

-Hydrophobic chromatography

-Affinity chromatography

GEL FILTRATION

This method is also known as size exclusion chromatography, as it separates proteins on the basis of their molecular mass related to their size.

GEL FILTRATION

These gels are prepared from agarose, acrylamide, and dextran polymers.

Ion-exchange chromatography:

cation or anion is attached to resin beads, depending upon the electric property of proteins.

Ion-exchange chromatography:

-If the desired protein is -vely charged then +ve charged resin beads are used.

-The resin beads is packed in the column.

Hydrophobic chromatography:

This chromatography was developed to purify proteins by exploiting their surface hydrophobicity.

Affinity chromatography:

a compound having specific affinity to desired protein is attached to

the resin. For. e.g. Antibody against desired protein is coated on resin.

Affinity chromatography:

-All the other protein gets eluted.

-Only the undesired protein gets eluted.

Size Determination

Size exclusion chromatography or SDS PAGE comparison with an expected size of protein (known e.g. from reference or cDNA)

Western Blotting

-Binding to the specific primary antibody, detected via labeled or enzymatically active secondary antibody

Biochemical assays in native gels

IdentificatiMass Spectrometryon of enzymes by their characteristic activity Identification of metalloproteins by their metal content

Mass Spectrometry

-Fragmentation of the protein, identification of fragment sizes, and subsequent comparison to a library of known fragmentation patterns

N-terminal Sequencing (Edman degradation)

-Sequential chemical removal of individual amino acids from the N-terminus

APPLICATIONS OF PURIFIED PROTEINS

-Hormones – Insulin, Erythropoietin, Clotting factors

-Diagnostics – LFT, KFT, TFT

-Cellular markers - Cancer

-In recombinant DNA technology to locate gene of interest

-Vaccines

-Supplements

Six common medical applications of purified proteins are:

-diabetes drugs.

-weight-loss drugs.

-diagnostics.

-vaccines.

-antibody-based drugs.

-waterproof adhesives.

Insulin

the protein insulin helps to regulate sugar or glucose levels.

Insulin

was originally purified from dog pancreas in the 1920s, and other animal

sources was used for decades to treat diabetics

Semaglutide

is a peptide drug that is used to treat type 2 diabetes.

Semaglutide

It consists of 26 amino acids that mimic the natural hormone glucagon-like peptide-1 (GLP-1). GLP-1 is released in response to food intake and stimulates insulin production.

Semaglutide

functionally uncouples the feeling of satiety (or fullness) from having

eaten, allowing people to eat less and lose weight.

GLP-1

reduces appetite, and in that way acts as a feedback loop to our brain -

signaling that we have had enough to eat and can stop.

DIAGNOSTICS

allowed us to determine if we were infected with certain diseases.

VACCINES

used live or inactivated pathogens to train our immune systems to

recognize potential invaders and rapidly tamp down future infections.

VACCINES

deliver the mRNA message molecule that encodes for pathogenic proteins.

ANTIBODIES

Our body produces…as part of the immune response to vaccines and

pathogens.

ANTIBODIES

recognize and bind tightly to pathogens and their molecules to alert our

immune system that clean-up is needed.

WATERPROOF ADHESIVES

are useful wound-healing devices but have limited utility in wet

environments, thereby their effectiveness on skin is depleted when wet, and that they are essentially useless inside the body where blood and other bodily fluids are plentiful.

It’s thought that these novel adhesives will have key roles including:

-surgical tissue glues to repair bleeding, tissue perforations, and fistulas.

-promoting heart regeneration after myocardial infarction.

-aiding tissue grafts for treating hernias, ulcers, and burns.

ENZYME

ENZYME

are a very important type of macromolecular biological catalysts. 

ENZYME

is a protein or RNA produced by living cells, which is highly  specific and highly catalytic to its substrates. 

ENZYME

can be defined as biological polymers that catalyze biochemical  reactions.” 

Catalysts

-speed up chemical reaction 

 ENZYME

was coined in 1878  by Friedrich Wilhelm Kuhne

Dubrunfaut (1830)

prepared malt extract from germinating barley seeds.

Payen and Persoz (1833)

prepared an enzyme, the diastase (now known as  amylase), from malt extract 

Amylase

present in saliva

Horace de Saussure

prepared a substance from germinating wheat which acted  like diastase 

 Theodor Schwann

succeeded in extracting pepsin and later trypsin