TY  - THES
AU  - Sigurdsson, Gudmundur
TI  - Overexpression and Purification of SLPI, a proteaseinhibitor from Streptomyces Lividans, in E. coli
PB  - University of Iceland
VL  - Dissertation
M1  - PUBDB-2025-04020
SP  - 1-65
PY  - 2025
N1  - Dissertation, University of Iceland, 2025
AB  - AbstractThis project focuses on the overexpression and purification of Streptomyces lividansprotease inhibitor (SLPI) in transformed E. coli to compare the efficiency of two methods ofexpression induction. SLPI belongs to the Streptomyces subtilisin inhibitor (SSI) proteinfamily, naturally found in S. lividans. Those methods are the IPTG method and autoinductionmethod. A cloned pET vector containing genomic DNA for SLPI will be transformed intoLemo21 E. coli cells, overexpressed, and then purified through a five-step process, includingsalt precipitation and column purification. The total amount of protein and SLPI producedusing either method will be compared using several analyses. Each step of the purificationprocess for both methods will be analyzed using a Bradford assay to determine proteinconcentration. SDS-PAGE will be run on each step to verify concentration results for thepresence of a protein of SLPI’s size and its purity. SLPI presence will also be verifiedthrough a serine protease (VPR) inhibitor activity assay in the presence of a color-changingsubstrate sample. The characteristics of the isolated protein at the end of purification will bemeasured using CD to determine the mean residue ellipticity (MRE) and melting point (TM).This was compared to the reference MSc thesis and showed that SLPI was isolated.Additionally, SAXS characterization of SLPI will be used. Overexpression of SLPI usingthe autoinduction method was more efficient than the IPTG method, primarily due tofacilitating a richer and more optimized expression environment for the E. coli culture thanthe manual IPTG induction method.
LB  - PUB:(DE-HGF)11
UR  - https://bib-pubdb1.desy.de/record/638202
ER  -