Characteristics of elastase strain K-Repeat-In-Toxin fusion protein overexpressed from newly constructed genetic tools
<p>This study was intended to construct a new Escherichia coli-Pseudomonas shuttle</p><p>vector for overexpression of elastase strain K in both E. coli and Pseudomonas as well</p><p>as for rapid purification using new RTX-tag. A 6...
Saved in:
| Main Author: | |
|---|---|
| Format: | thesis |
| Language: | eng |
| Published: |
2021
|
| Subjects: | |
| Online Access: | https://ir.upsi.edu.my/detailsg.php?det=8635 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | <p>This study was intended to construct a new Escherichia coli-Pseudomonas shuttle</p><p>vector for overexpression of elastase strain K in both E. coli and Pseudomonas as well</p><p>as for rapid purification using new RTX-tag. A 6.5 kb novel shuttle vector, designated</p><p>as pSIT/RTX, was constructed from pCon2(3) as to improvise the expression of</p><p>pCon2(3). pSIT/RTX was employed with tightly regulated promoter PT7(A1/O4/O3) for</p><p>controlling gene expression, stabilizing fragment (SF) for replication and maintenance</p><p>of plasmid in E. coli and P. aeruginosa, attB gene for genome integration, elastase</p><p>strain K as passenger enzyme and RTX-tag which is located at C-terminal for rapid</p><p>purification. E. coli TOP10/pSIT/RTX was chosen to proceed with purification as the</p><p>highest amount of proteolytic activity was detected at 12 h after incooperation with 0.6</p><p>mM IPTG (Isopropyl - d-1-thiogalactopyranoside). Elastase strain K-RTX fusion</p><p>protein was purified using Ca2+ as novel ligand in immobilized-metal affinity</p><p>chromatography (IMAC) with 28 % recovery and 3.8 fold. The estimated molecular</p><p>weight as observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis</p><p>(SDS-PAGE) was 73 kDA, with optimal temperature and pH were 40oC and pH6,</p><p>respectively. The proteolytic activity was significantly enhanced by increasing the</p><p>concentration of Na+ and Cu2+ ions and more stable in phenylmethylsulfonyl fluoride</p><p>(PMSF), Tween20 and Triton-X-100. On the downside, Ni2+, Zn2+, n-dodecane, ntetradocane,</p><p>dithiothreitol (DTT) and SDS showed strong inhibition on the proteolytic</p><p>activity. Elastase strain K exhibited preference towards 25% (v/v) of Dimethyl</p><p>sulfoxide (DMSO), methanol and pyridine as their uniqueness as an organic solvent</p><p>tolerant enzyme. Congo-red as the most specified substrate for elastase recorded the</p><p>lowest release of its product. As a concluding remark, experimental work conducted in</p><p>this study had indeed highlighted several achievements, novelties and findings</p><p>including construction of vectors which had led to the overexpression of elastase strain</p><p>K by constructed vectors, the using of RTX-tag for purification via IMAC and most</p><p>importantly, the remarkable stability of elastase strain K in hydrophilic organic</p><p>solvents.</p> |
|---|