基因,蛋白,免疫 课程之蛋白组学 2.doc

This lecture covers different aspects of the production of recombinant proteins. Recombinant proteins are made because it is often difficult to purify sufficient quantities of proteins, especially human proteins from natural sources. Recombinant proteins are used to study the activity and function of proteins. An integral component of these studies is knowledge of the structure of proteins and recombinant proteins have proved invaluable far structural studies. Recombinant proteins are used to raise antibodies with which to analyse the naturally occurring proteins. Recombinant proteins are used increasingly widely in industry and in medicine. There is a plethora of different expression systems with which to make recombinant proteins. Recombinant proteins can be expressed and regulated in cells in which their activity is to be studied. Learning objectives: By the end of your revision, you should be aware of the different ways in which recombinant proteins are produced. You should understand the relative merits of different expression systems and the considerations that might lead you to chose one rather than another. Background reading: Gene Cloning and Manipulation C. Howe, Chapters 8 and 9 Recombinant DNA JD Watson, M. Gilman, J. Witkowski, M. Zoller Chapter 12 pp213-226 and Chapter 23 pp453-460  Main steps involved Construction of expression vector In most instances a commercially available vector will be used This will contain the following features: Promoter This should be strong, but not too strong especially if it is to be inducible possibly inducible Translation initiation and termination signals For instance, an E.coli ribosome binding site (GAGG)or a Kozak sequence around ATG Sequences to determine the cellular location of recombinant protein For instance for secretion Fusion proteins In frame fusion sequences, at N- or C-terminus which can give stability. Useful for purification but must then consider subsequent cleavage Tags Short fusion sequences