基因,蛋白,免疫 课程之蛋白组学 3.doc

This lecture covers different ways in which in vitro mutagenesis is achieved. In in vitro mutagenesis, the base sequence of DNA is altered to test the effect of the change on the function of the gene. In vitro mutagenesis is of academic value to dissect the function of individual residues, to probe their structural role and to study interactions with small ligands, DNA and other proteins. In vitro mutagenesis is used in protein engineering to tailor a protein as closely as possible to its application. Unwanted traits may be removed or the activity of the protein can be improved eg affinity, specificity or in vivo performance. Learning objectives: By the end of your revision, you should understand what is involved in in vitro mutagenesis and different methods that can be used to create the desired protein analogues. You should appreciate the power of this technology in research and for industry. Background reading: Recombinant DNA JD Watson, M. Gilman, J. Witkowski, M. Zoller Chapter 11 pp 191-209 and Chapter 23 pp 465-468 Protein structure C Branden, J Tooze Chapter 11 pp191-209 and Chapter 13 pp 465-468 http://www.queensu.ca/micr/micr436/notes/436-mutagenesis/sld001.htm (slides 1-30) Behncken S.N., Waters M.J. (1999) Molecular recognition events involved in the activation of the growth hormone receptor by growth hormone. J. Mol. Recognition 12:355-362.  Main steps involved Have expression system Mutation of target sequence . Isolation of mutated plasmids Expression ( purification of protein analogue Verification or identification Of DNA by: sequencing Of protein by: sequencing laser desorption mass spectroscopy structural analyses eg: CD, NMR, X-ray Analysis Of the phenotypic consequences of the DNA mutation on the structure or function of the resulting protein analogue  III. Random mutagenesis Restriction endonuclease sites provide access for mutagenesis Generate blunt end S1 nuclease Fill in Klenow + dNTPs Linker insertion Insert linker ( Res