Date of Award
David A. Johnson
Thesis Professor Department
Biochemistry and Molecular Biology
Human neutrophils are the most abundant type of white blood cell and provide the body with a line of defense against foreign, infectious microorganisms. Contained within the azurophilic granules in the cytoplasm of neutrophils are three serine proteases, Human Neutrophil Elastase, Cathepsin G, and Protease 3. Once a foreign bacterium is engulfed by white blood cells, these enzymes attack and degrade the invading body, thus killing it (Reeves et al., 2002). The focus of this research is centered on the production of one of the serine proteases, human neutrophil elastase (HNE), and while the importance of HNE can be seen, genetic mutations or improper regulation can compromise a person’s immunity. Neutropenia (a low neutrophil count) is one such disease caused by a genetic mutation of HNE that results in susceptibility to infection (Li and Horwitz, 2001). Additionally, HNE is a powerful enzyme that can attack the elastin of the lung if not properly controlled. Consequently, genetic deficiencies of alpha-1 proteinase inhibitor protein in the blood can result in emphysema because active HNE released from neutrophils is free to degrade lung tissue (Laurell and Eriksson, 1965).
Recombinant HNE is not currently available, and the enzyme must be isolated from human blood cells, which has inherent hazards. Additionally, the lack of recombinant HNE has prevented studies involving site–directed mutagenesis to study the intracellular processing of HNE near its C-terminal end where mutations have been found to result in neutropenia. Kinetic studies of the full-length HNE might shed some light on why its C-terminal region is removed before storage in cytoplasmic granules.
The HNE DNA sequence was first codon optimized for yeast and commercially synthesized. It was then fused with DNA for eGFP (enhanced green fluorescent protein) via an enterokinase cleavage site (D4K). This DNA construct (eGFP-D4K-HNE) was then inserted into the Kluyveromyces lactis (K. lactis) pKLAC1 vector, downstream of the alpha mating factor which directs proteins for secretion. Then, chemically competent GG799 cells (a strain of K. lactis) were transformed with the linearized pKLAC1-eGFP-D4K-HNE insert through a protocol from New England Biolabs. Theoretically, the gene integrates into the yeast genome upon transformation via sequences within the pKLAC1 vector that are homologous with the LAC4 gene promoter that allows for galactose utilization (Colussi 2005). Acetamide was used as a selectable marker because wild type K. lactis cells are not able to use acetamide as a nitrogen source. The pKLAC1 vector, however, contains the Aspergillus nidulans gene acetamidase (amdS) that allows only transformants to grow on plates with acetamide as the sole nitrogen source (Read 2007).
Selected colonies were transferred to both liquid and agar-based synthetic media with galactose to induce transcription and translation of the HNE gene to produce the eGFP-D4K-HNE fusion, and screened via fluorescence microscopy for production of eGFP. None of the screened colonies tested positive for the presence of the fusion protein.
Honors Thesis - Open Access
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Klimecki, Haley M., "Expression of Human Neutrophil Elastase in K. Lactis." (2010). Undergraduate Honors Theses. Paper 2. https://dc.etsu.edu/honors/2
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