Kendriya Vidyalaya No. 1 Salt Lake
Class XII - Monthly Test -2015-16
Subject: Biotechnology (Set 1)
Max. Marks – 40
Time: 1 ½ hours
Section A
(1x5=5)
1. Name a human disease caused by: abnormal proteins and lack of protein.
A human disease caused by abnormal proteins – sickle cell anaemia due to abnormal
beta chain of haemoglobin
A human disease caused by lack of proteins – SCID (severe combined immuno
deficiency) caused by absence of ADA gene
2. Why is absorbance at 280nm used to monitor the protein concentration during
purification?
This is because taking absorbance at 280nm is an easy, fast and non-destructive
procedure for monitoring protein concentration during purification.
3. Why is whey considered a nutraceutical protein?
Whey is a nutraceutical protein because it has both nutritional and therapeutic
functions. Whey is rich in BCAAs, has a high biological value and high PER. It also
results in elevation of a tripeptide glutathione which has a range of beneficial
functions like detoxification of xenobiotics.
4. How can you confirm that a serine residue is involved in the catalysis of an enzyme?
The presence of a serine residue in the catalysis of an enzyme can be confirmed by the
addition of organophosphate compounds which specifically react with acidic serine
residues and knock off protein activity.
5. Why is agitation avoided during the purification of proteins?
Agitation causes the denaturation of proteins and hence has to be avoided during the
purification of proteins.
Section B
(2x5=10)
6. What is the role of hydrophobic interactions in the structure of proteins?
Water molecules form an extensive network by the formation of hydrogen bonds.
Non-polar or hydrophobic molecules cannot form hydrogen bonds or have
electrostatic interactions with water molecules. Thus when these molecules are added
to water, water forces these molecules out of solution to minimize the surface of
contact and minimize the number of hydrogen bonds which are broken. with interact
with water molecules and are therefore forced out of this network. Thus hydrophobic
interactions are a manifestation of hydrogen bonding network in water.
Hydrophobic forces are the most important in driving proteins to fold into compact
structures in water. In proteins, hydrophobic regions are preferentially located away
from the surface of the molecule and form the interior core of the protein.
7. Which of the following proteins would be expected to migrate faster in SDS-PAGE
and why?
PROTEIN
MW (Da)
a. α-Macroglobulin
820000
b.
c.
d.
e.
Lysozyme
Serum albumin
Retinol binding protein
α-antitrypsin
15,000
69,000
21,000
450000
The protein that would migrate faster in the SDS-PAGE would be lysozyme. The
technique seperates molecules based on molecular weight. Molecules with lower
molecular weight would travel faster in the gel than the molecules with higher molecular
weight. Since lysozyme has the least molecular weight among the given proteins, it can
be expected to travel fastest.
8. What do you mean by aqueous two-phase partition process for separation of proteins?
Aqueous two-phase partition process is an effective separation technique to separate
cellular debris from soluble-proteins. When a crude cell homogenate is added to a
biphasic mixture of dextran and polyethylene glycol, the cellular debris partitions to
the lower, more polar and dense phase, dextran. The soluble proteins remain in the
upper PEG phase.
Diagram on page 42, fig. 8.
9. Briefly describe the development of any two protein-based products.
Therapeutic antibodies and enzymes: Monoclonal antibody preparations as well as
antibody fragments produced by recombinant DNA technology have been used for
therapeutic purposes. OKT-3 is a monoclonal antibody that is used to prevent
rejection following kidney transplantation because the antibody blocks those immune
cells which attack foreign grafts.
Industrial enzymes: Many proteolytic enzymes have been used for various industrial
purposes in beverage, detergent, bread, leather, and meat industries. Alcalase is an
enzyme used in the soap industry while papain is used in the beverage industry.
10. What is OKT3? Why is it used?
OKT-3 is a monoclonal antibody. It is used to prevent rejection following kidney
transplantation because the antibody blocks those immune cells which attack foreign
grafts.
Section C
(3x5=15)
11. Discuss the use of designing a protein taking a suitable example.
Engineering of proteins is used to improve the properties of proteins. It is often
required to increase the stability of proteins when exposed to harsh conditions like
high temperature or organic solvents that are encountered in industrial processes. One
of the best examples of protein designing is the protease subtilisin. Subtilisin is a
protease (27kD) produced by bacteria and can digest a broad range of proteins that
soil clothing. The enzymatic activity of subtilisin is contributed to the catalytic triad,
i.e Ser221, His64, and Asp32. The native enzyme is easily inactivated by bleach due
to oxidation of the amino acid residue Methionine222 in the protein. Using sitedirected mutagenesis of the subtilisin gene in E.coli, the methionine was substituted
by a variety of other amino acids and the enzyme activity measured in the presence of
bleach. It was observed that substitution of Met222 with Ala222 was the best in terms
of activity and stability. Nowadays, many laundry detergents contain cloned,
genetically engineered subtilisin.
12. What is molecular pharming? Mention any four advantages of producing recombinant
proteins in milk.
Molecular pharming is the technology of producing pharmaceuticals using genetically
modified plants or animals.
Advantages are:
i)
High production capacity
ii)
Ease of source material collection
iii)
Moderate capital instrument requirements and low operational cost
iv)
Ease of production including purification and scale-up
13. Briefly indicate the steps involved in protein fingerprinting.
Protein fingerprinting involves the generation and 2-D analysis of peptides from a
protein. The steps are:
i)
Pure Hb and scHb are taken separately and digested with the proteolytic
enzyme trypsin which cleaves the protein after basic amino acids Arg and Lys.
ii)
Two separate strips of whatman filter paper are spotted with Hb and scHb
tryptic peptides and are separated using the technique of paper electrophoresis
at pH 2. Highly charged peptides migrate more towards the anode/cathode.
iii)
The paper strips are dried and chromatographed at right angles using the
solvent butanol:water:acetic acid. Here separation occurs based on the
partition coefficient between solvent and paper which is dependent on relative
hydrophobicity of peptides.
iv)
The chromatogram is visualized by using suitable stains and the peptide map
obtained is compared to locate the differences.
Diagram: Fig 6 on page 37
14. What is downstream processing? What strategy would you adopt to purify a
recombinant protein that is secreted into the growth medium?
Downstream processing refers to the processes that are involved in the recovery and
purification of useful products from a fermentation medium or other industrial
processes.
The strategy to purify a recombinant protein secreted into the growth medium is
shown in the flowchart:
Microbial source
Fermentation
Extra cellular Protein
Removal of cells
Purification of the protein
(preciitaion, ion exchange, gel
permeation chromatography,
affinity chromatography
Incorporation of stabilizers,
preservatives etc. adjustment to
require biopotency
Concentration of purified
extract
(precipitation/precipitation)
Final product format
Solid (spray dry/ drum dry /
pelleting / encapsulation /
freeze dry)
liquid
15. An E.coli produces about 2000
molecules of an enzyme per cell. If we have to purify 1g of this intracellular protein,
estimate how many cells of bacteria would be required theoretically? The molecular
weight of this protein is 1,00,000.
1,00,000 g of the protein corresponds to 1 mol of enzyme which corresponds to 6.023
x 1023 molecules.
Hence 1g of enzyme has 1/1,00,000 x 6.023 x 1023 or 6.023 x 1018 molecules.
2000 molecules of the enzyme are present in one cell.
Therefore, 6.023 x 1018 molecules are present in 6.023 x 1018 / 2000 = 3.011 x 1015
cells.
Section D
(5x2=10)
16. ‘The shape of a protein determines its function’. Describe this statement giving a
suitable example.
The shape of a protein determines its function. This can be explained taking the
example of chymotrypsin. Chymotrypsin hydrolyses peptide bonds following bulky
aromatic amino acids in polypeptides. It is synthesized in the pancreas and through
the pancreatic duct released into the duodenum. It is made up of a linear chain of 245
amino acids and in the 3-D structure three amino acid residues his57, asp102, and
ser195 come close together in space to form a charge relay system. Asp102 hydrogen
bonds with adjacent his57 by partially borrowing the hydrogen ion from the latter. His
57 attracts a hydrogen ion from the adjacent ser195. Due to the unique constellation
of the three amino acid residues due to the folding of protein, ser195 becomes acidic.
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 hydrolyze the bond. The active site of chymotrypsin has a large space
lined by hydrophobic residues which allow only bulky aromatic, hydrophobic amino
acids to bind. This binding brings the susceptible peptide bond close to the attacking
ser195 residue.
Diagram: fig 5 on page 35
17. Describe the technique of mass spectroscopy using a suitable diagram. What is its
application in the study of proteins?
A mass spectrometer is an analytical device that determines the molecular weight of
chemical compounds by separating molecular ions according to their mass/charge
ratios.
The technique involves the introduction of a vapourized sample of a protein or
peptide intot he instrument wherein it undergoes ionization. The charged molecules
are then electrostatically propelled into a mass analyser which separates the ions
according to their m/z ratio. The signal received upon detection of the ions at the
detector is transferred to a computer which stores and processes the information.
Application: Mass spectrometry is an extremely useful technique to obtain protein
structural information such as peptide mass or amino acid sequences. It is also useful
in identifying the type and location of amino acid modification within proteins.
Diagram: fig 10 on page 45