Research@Lincoln
    • Login
     
    View Item 
    •   Research@Lincoln Home
    • Theses and Dissertations
    • Theses and Dissertations with Restricted Access
    • View Item
    •   Research@Lincoln Home
    • Theses and Dissertations
    • Theses and Dissertations with Restricted Access
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Construction, expression and characterisation of recombinant a1-antitrypsin mutants

    Elliott, Peter Ross
    Abstract
    Alpha1-antitrypsin is the archetypal member of the serine protease inhibitor (serpin) superfamily. It is synthesised and secreted in the liver and its prime function is to form complexes with and inhibit the enzyme neutrophil elastase. This enzyme, released by neutrophils, is able to digest elastin fibres in the lungs and if uncontrolled may give rise to the destructive lung disease emphysema. A number of variant α1-antitrypsin molecules have been characterised which contain clinically relevant mutations. Substitution of the P1 methionine residue (position 358) in the reactive centre loop changes the inhibitory specificity of the molecule and may reduce or enhance its capacity to inhibit neutrophil elastase. The Z mutation (342 glutamic acid to lysine) results in loop sheet polymerisation of antitrypsin and accumulation of polymers within hepatocytes to give reduced circulating plasma levels. This accumulation of antitrypsin in the liver predisposes the individual to cirrhosis and, the low circulating plasma levels predispose to emphysema. In an attempt to understand the effect these mutations have on the structure and function of the antitrypsin molecule, site directed mutants have been constructed in which the DNA coding for the P1 and P17 residues has been mutated to code for different amino acids. Mutants constructed include P1 arginine, leucine valine and P17 arginine, lysine, arginine, glutamine and histidine. These were then expressed as recombinant proteins in E.coli, using a 10 litre fermentation system, with a maximal biomass production of approximately 24g/l dry weight. The proteins were extracted from the cell mass, purified to homogeneity and partially characterised. Recovery of between 2 and 15 grams of recombinant antitrypsin per purification was obtained depending on the mutant expressed.... [Show full abstract]
    Keywords
    Alpha 1-Antitrypsin (A1AT); fermentation; mutagenesis; purification; Escherichia coli; recombinant; serine protease inhibitor; enzymes; A1AT deficiency; DNA; amino acids; mutation; purification; bleeding disorder
    Fields of Research
    1101 Medical Biochemistry and Metabolomics; 060101 Analytical Biochemistry; 060405 Gene Expression (incl. Microarray and other genome-wide approaches); 110202 Haematology
    Date
    1993
    Type
    Dissertation
    Access Rights
    Digital thesis can be viewed by current staff and students of Lincoln University only. If you are the author of this item, please contact us if you wish to discuss making the full text publicly available.
    Collections
    • Theses and Dissertations with Restricted Access [2370]
    • Department of Wine, Food and Molecular Biosciences [716]
    View/Open
    Staff/student login to read
    Share this

    on Twitter on Facebook on LinkedIn on Reddit on Tumblr by Email

    Metadata
     Expand record
    This service is maintained by Learning, Teaching and Library
    • Archive Policy
    • Copyright and Reuse
    • Deposit Guidelines and FAQ
    • Contact Us
     

     

    Browse

    All of Research@LincolnCommunities & CollectionsTitlesAuthorsKeywordsBy Issue DateThis CollectionTitlesAuthorsKeywordsBy Issue Date

    My Account

    LoginRegister

    Statistics

    View Usage Statistics
    This service is maintained by Learning, Teaching and Library
    • Archive Policy
    • Copyright and Reuse
    • Deposit Guidelines and FAQ
    • Contact Us