Cathepsin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG is a poorly understood enzyme, in part because it can only be obtained as mature enzyme purified from human blood, and because it seems to have dual specificity for chymotrypsin-like and trypsin-like substrates. Therefore, yeast Pichia pastoris was used to express immature recombinant human CatG to provide a source of the enzyme free of biohazards and to allow study of its dual specificity and C-terminal processing. To avoid potential cleavage by a yeast kexin protease, the amino sequence was modified without altering CatG’s biological activity to remove one glycosylation site and eight dibasic sites. An N-terminal 6-His-cytochrome B5 (CytB5) heme binding fusion domain was linked to the modified human CatG by an enteropeptidase cleavage site for activation. The DNA for this construct was codon-optimized and placed in the pPICzα secretion vector. After transforming P. pastoris strain X-33, 48 Zeocin-resistant clones were screened for relative levels of CatG activity following activation by recombinant human enteropeptidase. Recombinant human CatG was partially purified from fermentation medium by nickel affinity chromatography and its activity was confirmed by assays using the substrate Succinyl-Ala-Ala-Pro-Phe-SBzl. Supported by a Student Faculty Collaborative Grant from the ETSU Honors College and ETSU Office of Research and Sponsored Programs and by NHLB grant R15HL091770.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:honors-1238 |
| Date | 01 May 2014 |
| Creators | Perry, Evan T |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Undergraduate Honors Theses |
| Rights | Copyright by the authors., http://creativecommons.org/licenses/by-nc-nd/3.0/ |
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