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

  1. 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.

  1. 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.

  1. 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.

  1. 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)

  1. 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.

  1. Which of the following proteins would be expected to migrate faster in SDS-PAGE and why?

PROTEINMW (Da)

  1. α-Macroglobulin 820000
  2. Lysozyme15,000
  3. Serum albumin69,000
  4. Retinol binding protein 21,000
  5. α-antitrypsin450000

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.

  1. 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.

  1. 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.

  1. 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)

  1. 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 site-directed 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.

  1. 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

  1. 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

  1. 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:

  1. 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)

  1. ‘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

  1. 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