Download CHE-3H84 14-15 exam FINAL

UNIVERSITY OF EAST ANGLIA
School of Chemistry
Main Series UG Examination 2014-15
PROTEIN ENGINEERING
CHE-3H84
Time allowed: 2 hours
Answer THREE questions.
You are advised to spend an equal amount of time on each question.
All questions carry an equal number of marks.
Answer EACH question in a SEPARATE answer book.
The breakdown of marks within each question is indicated by the percentage figures
in brackets on the right.
Do not take this question paper out of the examinations room.
Notes are not permitted in this examination.
Do not turn over until you are told to do so by the Invigilator.
(CHE-3H84)
Module co-ordinator: Andrew Hemmings (BIO)
Copyright of the University of East Anglia
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1.
Answer ALL parts (a) to (e).
The sequence of a small section of a double stranded DNA molecule is:
5’-GATATCCTTTCGGCTGTCAGG-3’
3’-CTATAGGAAAGCCGACAGTCC-5’
Assume that the first codon starts in the first base of the DNA sequence above.
(a)
What is the mRNA sequence transcribed from this DNA sequence?
[10%]
(b)
What is the amino acid sequence of the protein encoded by this DNA
molecule? A codon table is presented below.
[10%]
(c)
How would the first amino acid residue in this region of the protein sequence
be changed to an alanine? Describe the full process, including the experimental
method.
[40%]
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(d)
The first six base pairs in the DNA sequence correspond to the recognition
site for the type II restriction enzyme EcoRV, which is a blunt end cutter.
(i)
Describe type II restriction enzymes, citing their main characteristics.
[10%]
(ii)
What is meant by blunt ended DNA, and what is the main advantage
and disadvantage of using a blunt end enzyme experimentally?
[10%]
(e)
DNA sequences such as the one above are obtained using one of the different
sequencing methods available. Briefly outline the principles of ONE of the latest
generation, ultra-high throughput sequencing methodologies.
[20%]
2.
Answer BOTH parts.
(a)
Protein engineering can be achieved by rational design, directed evolution, or
using a semi-rational approach. Describe TWO of these methods, summarising the
steps required.
[50%]
(b)
Discuss briefly some of the advantages and disadvantages of solid-phase
peptide synthesis. Include at least three of each.
[50%]
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3.
Answer BOTH parts.
(a)
Describe the transition state analogue approach in the design of artificial
enzymes for ester hydrolysis by molecular imprinting.
[60%]
(b)
tert-Butyloxycarbonyl (BOC) and tertiary butyl esters are commonly used as
protecting groups in solid phase peptide synthesis. Define the type of functional
group they are used to protect and the mechanism of their removal in the
deprotection steps.
[40%]
4.
Answer ALL parts (a) to (c).
Under the terms of a contract held with a life sciences company you must supply 10
mg of a highly purified recombinant engineered IgG antibody light chain (rIgG-L).
rIgG-L has a molecular weight of 25 kDa, a pI of 8.0 and has neither cofactors nor
disulfide bridges. An inducible vector, pIgG-L, is available for overexpression of rIgGL in the cytoplasm of Escherichia coli. Note that IgG antibody light chain proteins are
known to bind with high affinity to Protein L in buffered solutions above pH 7 and
that your contract states that the protein you supply must be at least 99.9 % (w/w)
pure.
(a)
Discuss the methodologies available for the fermenter-based, large scale
production of a cell-free lysate containing rIgG-L using Escherichia coli as a
heterologous expression host.
[30%]
(b)
Explain how you would produce a starter culture for a fermenter run, starting
from the glycerol stock of Escherichia coli bacteria and the pIgG-L vector.
[20%]
(c)
You have access to common chromatographic resins including a resin
containing beads to which native Protein L has been covalently attached. Describe a
possible purification strategy for the recombinant protein, explaining the physical
basis of each method involved and the considerations you have taken into account
in developing your strategy.
[50%]
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