Objective. Albumin is an abundant and slowly-cleared plasma protein. Our laboratory previously incorporated albumin into recombinant fusion proteins to extend the plasma half-life of small proteins of potential therapeutic utility. We sought to determine if reiteration of albumin in a single polypeptide chain would further extend its half-life. Design. Hexahistidine-tagged rabbit serum albumin (RSA) with Cys 34 altered to Ala to prevent disulphide-bonded dimerization was produced in yeast (H₆-RSA-C34A), and compared to a reiterated forma of H₆-RSA-C34A containing two domains of amino acids 1-584 separated by a hexaglycine spacer. Clearance of these purified iodinated proteins was compared in rabbits. Materials and Methods. Site-directed mutagenesis employing PCR was used to alter the encoding plasmid. Proteins secreted from transformed Pichia pastoris yeast cell lines were purified using nickel-chelated affinity chromatography and radioiodinated by the Iodogen method. Labeled proteins were injected intravenously into rabbits and the residual acid-precipitable protein concentration in serial plasma samples was determined over time. Results. P. 𝘱𝘢𝘴𝘵𝘰𝘳𝘪𝘴 cells transformed with the expression plasmids secreted 140 kDa H₆-diRSA and 70 kDa H₆-RSA-C34A proteins, which were purified to apparent homogeneity. Mean terminal catabolic half-lives (±SD) were 4.9 (±0.7) and 3.0 (±0.3) days for H₆-RSA-C34A and H₆-diRSA, respectively. Then values were 9 for H₆-diRSA and 12 for H₆-RSA-C34A. conclusions. H₆-diRSA was cleared from the circulation more rapidly than H₆RSA-C34A. We hypothesized that increased catabolism of the reiterated molecule could be due to an increased rate of cellular uptake and endocytosis of H₆-diRSA due to increased avidity for cellular binding sites. Non-reiterated albumin therefore appears to be optimal as a carrier protein for small recombinant blood products. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22677 |
| Date | 09 1900 |
| Creators | McCurdy, Teresa |
| Contributors | Sheffield, W.P., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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