Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Protein folding wikipedia , lookup
Protein design wikipedia , lookup
List of types of proteins wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Protein purification wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Western blot wikipedia , lookup
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 Version 1 2 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%] question 1 continues…/ (CHE-3H84) Version 1 3 question 1 continued…/ (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%] (CHE-3H84) TURN OVER Version 1 4 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%] END OF PAPER (CHE-3H84) Version 1