JAWAHAR VIDYA MANDIR SHYAMALI, RANCHISHYAMALI, …...called Molecular Pharming. Advantages of producing recombinant proteins 1. High production capacity. 2. Ease of source material

STUDY MATERIAL & ASSIGNMENT STUDY MATERIAL & ASSIGNMENT STUDY MATERIAL & ASSIGNMENT STUDY MATERIAL & ASSIGNMENT

CHAPTER II: PROTEIN STRUCTURE AND ENGINEERINGCHAPTER II: PROTEIN STRUCTURE AND ENGINEERINGCHAPTER II: PROTEIN STRUCTURE AND ENGINEERINGCHAPTER II: PROTEIN STRUCTURE AND ENGINEERING

IntroductionIntroductionIntroductionIntroduction totototo thethethethe WWWWorldorldorldorld ofofofof Proteins:Proteins:Proteins:Proteins:

Almost everything that occurs in the cell involves one or more proteins. Proteins provide structure, catalyze

cellular reactions, and carry out a myriad of other tasks.

contractile protein, defense protein and regulatory protein.

Proteins have the maximum diversity in function.

acids, the sizes and sequence combinations and variations of each protein leads to millions of unique 3

structures and thereby functions.

JAWAHAR VIDYA MANDIRJAWAHAR VIDYA MANDIRJAWAHAR VIDYA MANDIRJAWAHAR VIDYA MANDIR

SHYAMALI, RANCHISHYAMALI, RANCHISHYAMALI, RANCHISHYAMALI, RANCHI

STUDY MATERIAL & ASSIGNMENT STUDY MATERIAL & ASSIGNMENT STUDY MATERIAL & ASSIGNMENT STUDY MATERIAL & ASSIGNMENT –––– BIOTECHNOLOGY, CLASS BIOTECHNOLOGY, CLASS BIOTECHNOLOGY, CLASS BIOTECHNOLOGY, CLASS ---- XIIXIIXIIXII

CHAPTER II: PROTEIN STRUCTURE AND ENGINEERINGCHAPTER II: PROTEIN STRUCTURE AND ENGINEERINGCHAPTER II: PROTEIN STRUCTURE AND ENGINEERINGCHAPTER II: PROTEIN STRUCTURE AND ENGINEERING


lular reactions, and carry out a myriad of other tasks. There can be transport protein,

and regulatory protein.

Proteins have the maximum diversity in function. Although all proteins are made up of

the sizes and sequence combinations and variations of each protein leads to millions of unique 3

Page 1111 of 16161616

XIIXIIXIIXII


There can be transport protein, storage protein,

Although all proteins are made up of 20 different amino

the sizes and sequence combinations and variations of each protein leads to millions of unique 3-D

Structural organization of proteinStructural organization of proteinStructural organization of proteinStructural organization of protein

Covalent and Non covalent interaction in proteiCovalent and Non covalent interaction in proteiCovalent and Non covalent interaction in proteiCovalent and Non covalent interaction in protei

Covalent bond- Peptide bond and disulfide bond

Non covalent bond---- The non-covalent interactions

can be broadly divided into four categories:

• • • • Ionic bonds

• • • • Hydrogen bonds



Protein molecules adopt a

specific three

conformation. This

structure is called

native fold. The native fold

has a large number of

favourable

within the protein.

Structural organization of proteinStructural organization of proteinStructural organization of proteinStructural organization of protein

Covalent and Non covalent interaction in proteiCovalent and Non covalent interaction in proteiCovalent and Non covalent interaction in proteiCovalent and Non covalent interaction in proteinsnsnsns:

Peptide bond and disulfide bond.

interactions involved in organising the structure of

categories:

Page 2222 of 16161616

Protein molecules adopt a

specific three-dimensional

conformation. This

structure is called the

native fold. The native fold

has a large number of

favourable interactions

within the protein.

of protein molecules

• • • • Van der Waals forces

• • • • Hydrophobic interactions

Determining the structuDetermining the structuDetermining the structuDetermining the structure of protein:re of protein:re of protein:re of protein:

1.1.1.1. Chemical Structure – Deducing the amino acid sequence by

• FDNB(1-Fluoro-2,4-dinitrobenzene

• Edman’s Reagent (phenylisothiocyanate;PITC)

2.2.2.2. 3D Structure – Understanding the protein folding in s

Magnetic resonance.

StructureStructureStructureStructure----FFFFunctionunctionunctionunction relationshiprelationshiprelationshiprelationship inininin ProteinsProteinsProteinsProteins

Shape of protein is paramount to the function

the oxygen carrying protein, haemoglobin,

Chymotrypsin,Chymotrypsin,Chymotrypsin,Chymotrypsin, aaaa proteolyticproteolyticproteolyticproteolytic enzymeenzymeenzymeenzyme

Chymotrypsin, hydrolyses peptide bonds

actually synthesised in the pancreas and

amino acid side residues, His57, Asp102 and Ser195 participate in the catalytic reaction by forming

a charge relay systemcharge relay systemcharge relay systemcharge relay system....



Deducing the amino acid sequence by Sequencing reagent:

dinitrobenzene) DEVELOPED BY DR. FREDERICK SANGER

phenylisothiocyanate;PITC) - DEVELOPED BY PEHR EDMAN

ing the protein folding in space by X-ray Crystallography and Nuclear

ProteinsProteinsProteinsProteins

function of a protein. Here are two proteins- an enzyme,

haemoglobin, to emphasise the importance of protein structure

bonds following bulky aromatic amino acid residues

and through the pancreatic duct released into the duodenum.

His57, Asp102 and Ser195 participate in the catalytic reaction by forming

The negatively charged Asp102 is able to hydrog

with the adjacent His57 partially borrowing the hydrogen

ion from the latter. The His57 makes good its partial

hydrogen ion loss to aspartate by attracting a hydrogen ion

from the adjacent Ser195 through the

like a relay race where the baton is passed from one

member to another, the difference here being that the

baton is a charge. The negatively charged oxygen anion is

able to make a nucleophilic attack on the carbonyl carbon

of the peptide bond of its substrate, loosening it so

water molecule can hydrolyse the bond

Page 3333 of 16161616

ray Crystallography and Nuclear

enzyme, chymotrypsin and

structure in its function.

residues in polypeptides is

into the duodenum. Three

His57, Asp102 and Ser195 participate in the catalytic reaction by forming

sp102 is able to hydrogen bond

is57 partially borrowing the hydrogen

is57 makes good its partial

hydrogen ion loss to aspartate by attracting a hydrogen ion

er195 through the His57 residue much

re the baton is passed from one

member to another, the difference here being that the

baton is a charge. The negatively charged oxygen anion is

able to make a nucleophilic attack on the carbonyl carbon

of the peptide bond of its substrate, loosening it so that a

water molecule can hydrolyse the bond.

MolecularMolecularMolecularMolecular DiseaseDiseaseDiseaseDisease---- SickleSickleSickleSickle cellcellcellcell aaaanaemianaemianaemianaemia

Red blood cells contort into a sickle shape. The cells die early, leaving a shortage of healthy red blood

Linus Pauling predicted that the sickled cell haemoglobi

amino acid. This was confirmed by V. M.

finger printing in the famous Laboratory of Molecular Biology (LMB)

Protein fingerpriProtein fingerpriProtein fingerpriProtein fingerprinting or Peptide Mappingnting or Peptide Mappingnting or Peptide Mappingnting or Peptide Mapping

This technique involves the generation

useful technique to compare similar proteins

The protein fingerprinting data has

electrophoresis patterns of entire proteins from



Red blood cells contort into a sickle shape. The cells die early, leaving a shortage of healthy red blood

auling predicted that the sickled cell haemoglobin differed from normal haemoglobin

M. Ingram in 1957 that pioneered a useful technique called protein

finger printing in the famous Laboratory of Molecular Biology (LMB) at Cambridge, UK.

nting or Peptide Mappingnting or Peptide Mappingnting or Peptide Mappingnting or Peptide Mapping

and 2-D analysis of peptides from a protein. P

proteins from different sources.

Protein fingerprinting

has been further augmented with new databases

proteins from a given cell type, a technique developed by O'Farrel.

Page 4444 of 16161616

Red blood cells contort into a sickle shape. The cells die early, leaving a shortage of healthy red blood cells.

from normal haemoglobin in a charged

a useful technique called protein

Peptide mapping is a

databases containing 2-D

O'Farrel.

2222----DDDD GelGelGelGel ElectrophoresisElectrophoresisElectrophoresisElectrophoresis

Two-dimensional gel electrophoresis (2DE) is the classical method

charge (isoelectric focusing, IEF) and of their size (sodium dodecyl sulfate polyacrylamide gel

electrophoresis, SDS-PAGE).

PurificationPurificationPurificationPurification ofofofof ProteinsProteinsProteinsProteins

The first step in determining the structure

Isolation of a protein from a microbial culture, plant and animal sources involves various separation

techniques. These steps are collectively known

In spite of a large biodiversity of microbes we are restricted to certain bacteria/organisms which can be used

as a source of protein as well as for introducing genes. These microorganisms are designated as "generally

regarded as safe" (GRASGRASGRASGRAS). GRAS listed organisms are non

produce any antibiotics.

The exact details of the purification scheme



dimensional gel electrophoresis (2DE) is the classical method to separate proteins on the basis of their


structure of a protein is to isolate it in a pure form from


collectively known as downstream processing.

versity of microbes we are restricted to certain bacteria/organisms which can be used


). GRAS listed organisms are non-pathogenic, non-toxic and generally should not

scheme for any given protein will depend upon a number

Page 5555 of 16161616

to separate proteins on the basis of their


its cellular location.


versity of microbes we are restricted to certain bacteria/organisms which can be used


toxic and generally should not

number of factors such as:

1. Exact source material chosen and

2. Quantity of protein required and

3. Physical, chemical and biological

All efforts should be made to maximise protein

which may be followed are:

1. Maintenance of a specific pH value range

2. Maintenance o f physiological conditions

3. Use of inhibitors to prevent the action of proteolytic enzymes.

4. Avoidance of agitation or addition

5. Minimise processing time

MOLECULAR PHARMINGMOLECULAR PHARMINGMOLECULAR PHARMINGMOLECULAR PHARMING

AqueousAqueousAqueousAqueous twotwotwotwo----phasephasephasephase partitionpartitionpartitionpartition



and location of the target protein (intracellular

hence amount of raw material processed.

biological properties of the protein

protein stability during various steps. Some of the general conditions

range of buffered solutions in which a protein is maximally

physiological conditions (% CO2 for animal cell culture and temperature).

e of inhibitors to prevent the action of proteolytic enzymes.

of chemicals which may denature the target protein.

Transgenic animals are created by direct micro

of DNA into ova or stem cells and produce

proteins in milk on a commercial scale. This

called Molecular Pharming.

Advantages of producing recombinant proteins

1. High production capacity.

2. Ease of source material collection (milking

3. Moderate capital instrument requirements

operational cost.

4. Ease of production including purification

Page 6666 of 16161616

or extracellular).

general conditions

is maximally stable.

temperature).

target protein.

direct micro-injection

produce insulin and other

scale. This technology is

proteins in milk are:

(milking cows).

requirements and low

purification and scale-up.

Aqueous two-phase partition is a liquid-

great potential for the extraction, separation, purification and enrichment of proteins and other soluble

materials. When a crude cell homogenate is added to a biphasic mixture of dextran and p

(PEG) the cellular debris partitions to the lower, more polar and dense phase, dextran. Separation of the two

phases achieves effective separation of cellular debris from soluble protein

ChaChaChaCharrrracterisationacterisationacterisationacterisation ofofofof ProteinsProteinsProteinsProteins

Techniques listed below characterise proteins

amino-acid sequence etc.

1. Electrophoretic techniques, SDS/PAGE.

2. Fingerprinting.

3. Two dimensional gel electrophoresis.

4. Protein sequencing.

5. Mass spectrometry

MassMassMassMass spectrometryspectrometryspectrometryspectrometry

Mass spectrometry (MS) has emerged as an important tool in biotechnology. It is extremely useful in

obtaining protein structural information such as peptide mass or amino acid sequences. It is also useful in



-liquid fractionation technique and has gained an interest because of


When a crude cell homogenate is added to a biphasic mixture of dextran and p

PEG) the cellular debris partitions to the lower, more polar and dense phase, dextran. Separation of the two

phases achieves effective separation of cellular debris from soluble protein.

proteins with respect to properties such as mass,

AGE.



Page 7777 of 16161616

fractionation technique and has gained an interest because of


When a crude cell homogenate is added to a biphasic mixture of dextran and polyethylene glycol

PEG) the cellular debris partitions to the lower, more polar and dense phase, dextran. Separation of the two

mass, isoelectric charge,



identifying the type and location of amino acid modification within proteins

Mass spectrometry (MS) analysis of proteins measures the mass

quantify molecules in simple and complex mixtures

as little as picomoles (10-12) of a protein

An outline of a mass spectromete

A sample M with a molecular weight greater

(M+nH)n+. Multiply charged ions on ana

can be deduced as also indicated in the legend

A protein with a molecular weight of 10,000 generates



and location of amino acid modification within proteins.

Mass spectrometry (MS) analysis of proteins measures the mass-to-charge ratio of ions to identify and

quantify molecules in simple and complex mixtures. One of the major attractions of mass

sample can be analysed.

An outline of a mass spectrometer

greater than 1200 D can give rise to multiple charged

analysis show patterns as indicated below from which

legend below.

A protein with a molecular weight of 10,000 generates five different peaks with the ions

Page 8888 of 16161616

charge ratio of ions to identify and

mass spectrometry is that

charged ions such as

which molecular mass

ve different peaks with the ions

containing 5, 4, 3, 2, and 1 charges, respecti

protein ions at 2001, 2501, 3334, 5001, and 10,001, respectively.

ProteinProteinProteinProtein BasedBasedBasedBased ProductsProductsProductsProducts

Proteins may be differentiated on the basis of their

sources.... From the commercial point of

following categories:

WheyWheyWheyWhey: Whey protein comprises 20 % of total milk

functional peptides, antioxidants and immunoglobu

glutathione (gamma-glutamyl cysteinyl

broad range of functions including detoxification

from the effect of oxygen intermediates and free radicals. More recently curd has also been

Blood products and vaccines

Therapeutic antibodies and enzymes

Therapeutic hormones and growth factors

Regulatory factors

Protein in analytical application

Industrial enzymes

Functional non-catalytic proteins

Nutraceutical proteins



containing 5, 4, 3, 2, and 1 charges, respectively. The mass spectrometer detects each of the

protein ions at 2001, 2501, 3334, 5001, and 10,001, respectively.

Proteins may be differentiated on the basis of their plant, animal and recombinant protein

of view, proteins based products may be classified

comprises 20 % of total milk protein and it is rich in branched and essential amino acids,

functional peptides, antioxidants and immunoglobulins. Whey proteins result in the elevation

cysteinyl glycine) in cells. This peptide is a reducing compound

detoxification of xenobiotics and protection of cellular c

from the effect of oxygen intermediates and free radicals. More recently curd has also been

Blood products and vaccines

Therapeutic antibodies and enzymes

Therapeutic hormones and growth factors

Protein in analytical application

catalytic proteins

Nutraceutical proteins

Page 9999 of 16161616

vely. The mass spectrometer detects each of the

animal and recombinant protein

classified into the

and it is rich in branched and essential amino acids,

elevation of a tripeptide

compound and has a

cellular components

from the effect of oxygen intermediates and free radicals. More recently curd has also been used as a pro-

biotic (administered with antibiotics) because

the intestinal tract.

WWWWheyheyheyhey protprotprotprot

FFFFunctionalunctionalunctionalunctional PropertyPropertyPropertyProperty ModeModeModeMode ofofofof actionactionactionaction

Whipping/Foaming Forms stable film

Emulsification Formation and

emulsions

Gelation Protein matrix

Viscosity Thickening, water

Water binding Hydrogen bonding

entrapment of

Solubility Protein solvation

Browning Undergoes Maillard

the amino groups

aldehyde groups

Flavour/Aroma Lactose reacts

DesigningDesigningDesigningDesigning ProteinsProteinsProteinsProteins (Protein(Protein(Protein(Protein Engineering)Engineering)Engineering)Engineering)

Protein engineeringProtein engineeringProtein engineeringProtein engineering is a method of changing a

change in the substrate specificity, or increased stability to the temperature, organic solvents, and/or

extremes of pH.

Stability in a folded protein is a balance between the stabilising (mainly hydrophobic) interactions and the

tendency towards destabilisation caused

unfolded form. The stability of a protein

favour stabilising interactions in a folded protein or destabilising inte



because it is a good source of beneficial bacteria which

protprotprotprotein: functional properties at a glanceein: functional properties at a glanceein: functional properties at a glanceein: functional properties at a glance

actionactionactionaction FFFFoodoodoodood SystemSystemSystemSystem

film Egg less cakes, desserts, whipped

and stabilization of fat Vegetarian sausages, salad

coffee whiteners, soups, cakes,

matrix formation and setting Meat, baked goods, cheeses

water binding Soups, gravies, salad dressings

bonding of water;

of water

Meats, sausages, cakes, breads

solvation Beverages

Maillard reaction (on heating,

groups of protein react with

groups of sugars)

Breads, biscuits, confections,

reacts with milk proteins Baked goods, biscuits, confectionaries,

sauces, soups, dairy products.

Engineering)Engineering)Engineering)Engineering)

is a method of changing a protein sequence to achieve a desired result, such as a



caused by the loss of conformational entropy as the protein

protein may however be changed by substituting amino

folded protein or destabilising interactions in an inactive protein.

Page 10101010 of 16161616

which can colonise

whipped topping

salad dressings,

cakes, infant

cheeses

dressings

breads

confections, sauces

Baked goods, biscuits, confectionaries,

products.

sequence to achieve a desired result, such as a



protein adopts the

substituting amino acids that either

ractions in an inactive protein.

Examples of protein engineering:

1.1.1.1. ImproImproImproImprovvvvinginginging laundrylaundrylaundrylaundry detergentdetergentdetergentdetergent SubtilisinSubtilisinSubtilisinSubtilisin

Subtilisin (27 kD) is a protease produced

soil clothing. The enzymatic activity of

Asp32.

The native enzyme subtilisin is easily inactivated

inactivation was due to oxidation of the amino

directed mutagenesis of the subtilisin

improved its activity and stability.

2.2.2.2. Creation of Novel ProteinsCreation of Novel ProteinsCreation of Novel ProteinsCreation of Novel Proteins

A recombinant vaccine based on

micromanipulation, unhindered supply and

novel synthetic gene has been assembled

Hepatitis B virus.

3.3.3.3. Improving nutritional value of cereals and legumesImproving nutritional value of cereals and legumesImproving nutritional value of cereals and legumesImproving nutritional value of cereals and legumes

Deficiencies in seeds of certain essential amino acids render the cereal grains or legumes unsuitable for a

balanced diet. Supplementation of diet with essential amino acids from other sources t

essential.

Whey protein is superior to other sources

val, lys and trp. The branched chain

proteins. They help in increasing the bio

absorbed by muscle cells for anabolic muscle building activity

BCAAs are released from the skeletal muscle; the carbon ske



SubtilisinSubtilisinSubtilisinSubtilisin

produced by bacteria that can digest a broad range of proteins

of subtilisin is contributed by a catalytic triad, i.e.,

inactivated by bleach (up to 90%). Careful studies

amino acid residue Methionine222 in the protein molecule. Using site

gene in E.coli, this methionine was substituted

selected epitopes may provide optimal design,

supply and safety needed for an effective vaccine. Working on these

assembled as a first step towards developing a subunit

Improving nutritional value of cereals and legumesImproving nutritional value of cereals and legumesImproving nutritional value of cereals and legumesImproving nutritional value of cereals and legumes

in seeds of certain essential amino acids render the cereal grains or legumes unsuitable for a

Supplementation of diet with essential amino acids from other sources t

sources of proteins especially with regard to branched amino

amino acids (BCAA) are essential for the biosynthesis

help in increasing the bio-availability of high complex carbohydrates intake and are

absorbed by muscle cells for anabolic muscle building activity .One of the theories is that during exercise the

BCAAs are released from the skeletal muscle; the carbon skeleton part is used as fuel and the nitrogen part is

Page 11111111 of 16161616

proteins that commonly

i.e., Ser221, His64 and

studies showed that this

protein molecule. Using site-

substituted by Ala222 and it

provide optimal design, scope for

orking on these lines, a

a subunit vaccine against

in seeds of certain essential amino acids render the cereal grains or legumes unsuitable for a

Supplementation of diet with essential amino acids from other sources therefore becomes

amino acids- ile, leu,

biosynthesis of muscle

availability of high complex carbohydrates intake and are

One of the theories is that during exercise the

leton part is used as fuel and the nitrogen part is

used to make alanine which then goes to the liver where it is turned into glucose for energy. So for athletes

who want to protect their existing mass, the idea is to take BCAA enriched foods before and af

BCAAs reduce muscle breakdown and

maintaining exercise performance and delaying

Important terms:

BiologicalBiologicalBiologicalBiological valuevaluevaluevalue (BV) measures the amount

of protein nitrogen that has been consumed. It

compared to rice, wheat, soya and egg proteins

ProteinProteinProteinProtein efficiefficiefficiefficiencyencyencyency rrrratioatioatioatio (PER) is used as a measure

consuming 1g of food protein. The PER value

milk, casein, soya, rice, wheat.

QUESTIONQUESTIONQUESTIONQUESTION 1111.Explain in brief any two types of non

ANSWER)ANSWER)ANSWER)ANSWER) 1. Ionic bonds

These involve interactions between the oppositely charged groups of a molecule. For example

charged amino acid side chains of lysine

chains of aspartate and glutamate. These

dominant bonds found in salts like sodium chloride wherein

with the negatively charged chloride ion.

2. Van der Waals forces

These forces are weak attractions (or repulsions)

types of forces are essentially contact forces

bonds can be important in macromolecules because

large total forces.




who want to protect their existing mass, the idea is to take BCAA enriched foods before and af

act as an energy source before and after exercise. Hence while

delaying exhaustion BCAAs are very important for

amount of protein nitrogen that is retained by the body from

consumed. It has been observed that the BV of whey proteins is the highest

compared to rice, wheat, soya and egg proteins.

measure of growth expressed in terms of weight gain

value of the following proteins are arranged in decreasing

QuestionsQuestionsQuestionsQuestions and Aand Aand Aand Annnnswersswersswersswers

Explain in brief any two types of non- covalent interactions found in proteins.

These involve interactions between the oppositely charged groups of a molecule. For example

lysine and arginine can form salt bridges with the negatively

glutamate. These ionic interactions are also known as salt bridges salt bridges salt bridges salt bridges

dominant bonds found in salts like sodium chloride wherein the positively charged sodium ion interacts

chloride ion.

repulsions) which occur between atoms at close range.

forces, proportional to the surface areas in contact.

macromolecules because the large surface areas involved can

Page 12121212 of 16161616


who want to protect their existing mass, the idea is to take BCAA enriched foods before and after excercise.

act as an energy source before and after exercise. Hence while

for muscle growth.

from a given amount

of whey proteins is the highest

gain of an adult by

decreasing order- whey,

covalent interactions found in proteins.

These involve interactions between the oppositely charged groups of a molecule. For example, the positively

the negatively charged side

salt bridges salt bridges salt bridges salt bridges because these are

sodium ion interacts

range. The Van der Waals

Though weak, these

can result in reasonably

QUESTIONQUESTIONQUESTIONQUESTION 2222. Describe the principle of MALDI technique. Write

ANSWER) ANSWER) ANSWER) ANSWER) A popular method called Matrix Assisted

volatalise and protonate peptides and proteins.

phase to a gas phase with the help of a solid

laser beam onto a sample suspended or dissolved in a matrix. The matrix plays a key role in this technique

by absorbing the laser light energy and causing the

plays a role in sample ionisation. The charged molecules are directed by electrostatic lenses from the

ionisation source to the mass analyzer

separating molecular ions according to their

USES: To identify the type and location of

information such as peptide mass or amino acid sequences

QUESTIONQUESTIONQUESTIONQUESTION 3333.Name a disease caused due to altered structure of protein?

ANSWER) ANSWER) ANSWER) ANSWER) Mad’s cow disease wherein the

prions.

QUESTIONQUESTIONQUESTIONQUESTION 4444. A protein from cell A and cell B is compared to find the

developed by O’Farrel. Name the technique. State its principle.

ANSWER) ANSWER) ANSWER) ANSWER) 2-D Gel Electrophoresis

Two different techniques are combined in

In IEF, proteins are separated on the basis

basis of their size and hence at the end of this electrophoretic run proteins are separated into 2

with high resolution as two properties

size.

QUESTIONQUESTIONQUESTIONQUESTION 5555.... An E. coli cell produces at



Describe the principle of MALDI technique. Write its two main uses in protein studies.

Matrix Assisted Laser Desorption Ionisation (MALDI)

proteins. In this procedure, the sample is transferred

solid matrix. Ion formation in MALDI is achieved by directing a pulsed


by absorbing the laser light energy and causing the matrix material to vaporise. In the gas phase, the matrix


ionisation source to the mass analyzer. This technique determines the molecular weight of prot

their mass/charge ratio (m/z) ratios.

of amino acid modification within proteins. To obtain protein structural

information such as peptide mass or amino acid sequences.

Name a disease caused due to altered structure of protein?

the disease itself appears to be propagated by infectious

A protein from cell A and cell B is compared to find the whole protein pattern by a technique

the technique. State its principle.

in this procedure- Isoelectro focussing (IEF) and SDS

basis of their different pI values. In SDS-PAGE proteins separate on the

hence at the end of this electrophoretic run proteins are separated into 2

of the proteins have been exploited in their separation

at least 2000 different proteins. One of these is our

Page 13131313 of 16161616

its two main uses in protein studies.

(MALDI) is used to

transferred from a condensed

Ion formation in MALDI is achieved by directing a pulsed


matrix material to vaporise. In the gas phase, the matrix


This technique determines the molecular weight of proteins by

proteins. To obtain protein structural

infectious proteins called

whole protein pattern by a technique

SDS-PAGE

AGE proteins separate on the

hence at the end of this electrophoretic run proteins are separated into 2-D patterns

separation- charge and

our enzyme of interest

produced at a level of 3000 molecules

intra-cellular enzyme, estimate how many

the molecular weight of the enzyme of

ANSWER) ANSWER) ANSWER) ANSWER) 1,00, 000 g of the protein of

6.023 x 1023 molecules (Avogadro no.)

Hence 1 g of enzyme has 1/1,00,000 x 6.023

3000 molecules of the enzyme are present

Therefore, 6.023 x 1018 molecules are present

QUESTIONQUESTIONQUESTIONQUESTION 6666 Write five parameters for

ANSWER) ANSWER) ANSWER) ANSWER) i) Maintenance of specific

ii) Maintenance of physiological

iii) Use of inhibitors to prevent

iv) Avoidance of agitation or

v) Minimize processing time

QUESTIONQUESTIONQUESTIONQUESTION 7777 Van der Waals forces are weak

ANSWER)ANSWER)ANSWER)ANSWER) Van der Waals forces are important in macromolecules such

surface areas involved can result in reasonably

the surface areas in contact. Several Van

QUESTIONQUESTIONQUESTIONQUESTION 8888 Using an example , describe

different sources.

ANSWER)ANSWER)ANSWER)ANSWER) Peptide mass fingerprinting

technique for protein identification in which

smaller peptides, whose absolute masses

MALDI-TOF or ESI-TOF. Mass spectrometry



per cell under optimum conditions. If we have

many cells of bacteria will be required theoretically?

of interest is 1,00,000.

of interest corresponds to 1 mole of enzyme which corresponds

6.023 x 1023 or 6.023 x 1018 molecules .

present in one cell.

present in 6.023 x 1018/3000 = 2.007 x 1015 cells.

for maximizing protein stability during purification

specific pH.

Maintenance of physiological conditions (%CO2 for animal cell culture and

prevent the action of proteolytic enzymes.

of agitation or addition of chemicals which may denature the

time

are weak attractions, but are important in proteins.

ls forces are important in macromolecules such as proteins

volved can result in reasonably large total forces. These are contact forces, proportional t

Van der Waals forces together give rise to stronger force

describe a technique commonly used to compare similar

fingerprinting (PMF) (also known as protein finger printi

which the unknown protein of interest is first

masses can be accurately measured with a mass spectrometer

spectrometry (MS) is an analytical technique that ionizes

Page 14141414 of 16161616

to purify 1g of this

theoretically? It is given that

which corresponds to

purification.

for animal cell culture and temperature)

the target protein.

proteins. Why?

proteins because the large

These are contact forces, proportional to

force.

compare similar proteins from

printing) is an analytical

first cleaved into

spectrometer such as

ionizes chemical species

and sorts the ions based on their mass-to

masses within a sample. Mass spectrometry

as well as complex mixtures. Example Normal

QUESTIONQUESTIONQUESTIONQUESTION 9999 Chymotrypsin catalyses the hydrolysis of proteins containing bulky, aromatic hydrophobic

amino acids. Comment.

ANSWER) Three amino acid side residues, His57, Asp102 and Ser195 participate in the catalytic reaction by

forming a charge relay system in chymotrypsin

nucleophilic attack on the carbonyl carbon of the peptide bond of its substrate. It loosens the carbonyl

carbon, so that a water molecule can hydrolyse the bond.

QUESTION 10QUESTION 10QUESTION 10QUESTION 10 If a given protein with a molecular weight of 20,000

is subjected to mass spectrometry, find the sequence of protein ions detected by the mass spectrometer.

ANSWER)

1. Why is sickle cell anaemia called "Molecular

2. Distinguish between chymotrypsinogen

3. E. coli is a rod shaped bacteria about 2

ml. Approximately 13.5% of the wet weight

particular enzyme per cell if the enzyme

soluble protein.



to-charge ratio. In simpler terms, a mass spectrum

spectrometry is used in many different fields and is applied

Normal Hb and Sc Hb. (diagram on page 4)

Chymotrypsin catalyses the hydrolysis of proteins containing bulky, aromatic hydrophobic

Three amino acid side residues, His57, Asp102 and Ser195 participate in the catalytic reaction by

charge relay system in chymotrypsin. The negatively charged oxygen anion is able to make a

carbon of the peptide bond of its substrate. It loosens the carbonyl

carbon, so that a water molecule can hydrolyse the bond. (diagram on page 3).

a given protein with a molecular weight of 20,000 daltons containing 5,4,3,2, and charges,

s subjected to mass spectrometry, find the sequence of protein ions detected by the mass spectrometer.

ASSIGNMENTASSIGNMENTASSIGNMENTASSIGNMENT

"Molecular disease"? How can sickle cell haemoglobin be identified?

chymotrypsinogen and chymotrypsin.

2 �m long and 1 �m in diameter. The average density of

weight of E. coli is soluble protein. Estimate the number

enzyme has a molecular weight of 100,000 and represents

Page 15151515 of 16161616

spectrum measures the

applied to pure samples

Chymotrypsin catalyses the hydrolysis of proteins containing bulky, aromatic hydrophobic

Three amino acid side residues, His57, Asp102 and Ser195 participate in the catalytic reaction by

The negatively charged oxygen anion is able to make a

carbon of the peptide bond of its substrate. It loosens the carbonyl

5,4,3,2, and charges,

s subjected to mass spectrometry, find the sequence of protein ions detected by the mass spectrometer.

be identified?

density of a cell is 1 .28 g/

number of molecules of a

represents 0.1 % of the total

4. Define subunit, domain, and quaternary

5. With an example, explain the development

6. Name two human diseases caused by the

7. a. What is the isoelectric point of a protein?

b. Write the principle of 2-D gel

c. Describe the aqueous two-phase

8. What is molecular pharming? Give four advantages of producing recombinant proteins in milk.

9. Specific activity increases during subsequent steps of a protein purification scheme. How can

you relate it with the purity of protein?

10. What will be the consequence if a protein is having an altered structure?

11. Give reasons for the following:

(i) Kappa casein is involved in micelle stabilization of milk proteins.

(ii)Whey protein detoxifies xenobiotics.

(iii) Curd is used as pro-biotic.

12. What is meant by tissue engineering? Explain any two important medical applications of tissue

engineering.

13. What is the hierarchical organization of protein structure? Indicate the nature of covalent and

non-covalent forces which determine the protein structure.

14. How can one use the method of aqueous

Also suggest various efforts which may be taken to maximize protein stability during such

separation.

15. In a variant of chymotrypsin, Asp 102 is replaced by Glu 102. Do you expect the enzyme to retain

activity? Schematically indicate the role of amino acid residues participating in catalysis.

Thalassemic patients produce excess alpha or b

binding capacity by their erythrocytes. How can it be determined as to which subunit is produced in excess?



quaternary structure in proteins.

development of one protein based product.

the absence of a protein.

protein?

electrophoresis.

phase partition process used for purification of proteins.


Specific activity increases during subsequent steps of a protein purification scheme. How can

protein?

What will be the consequence if a protein is having an altered structure?

(i) Kappa casein is involved in micelle stabilization of milk proteins.



forces which determine the protein structure.

14. How can one use the method of aqueous-two phase partitioning for the separation of proteins?




OR

Thalassemic patients produce excess alpha or beta subunits of haemoglobin leading to impaired oxygen


Page 16161616 of 16161616

of proteins.


Specific activity increases during subsequent steps of a protein purification scheme. How can



two phase partitioning for the separation of proteins?




eta subunits of haemoglobin leading to impaired oxygen-


Documents

JAWAHAR VIDYA MANDIR SHYAMALI, RANCHISHYAMALI, …...called Molecular Pharming. Advantages of producing recombinant proteins 1. High production capacity. 2. Ease of source material