Biomaterial Design: Human Tropoelastin Público
Pei, Yunyun (2010)
Abstract
Abstract
Biomaterial Design: Human Tropoelastin
By Yunyun Pei
Elastin is an important protein that provides connectivity and
elasticity in the
extracellular matrix. It is an extremely insoluble protein due to
the extensive
cross-linking at Lys residues. Tropoelastin is the soluble
precursor of elastin, and
therefore is of great importance for structure-function
relationship of elastin study.
The work described herein is focused on design and expression
of
recombinant human tropoelastin in E. coli. In this study,
five plasmids pJ246/STEL_1,
pJ246/STEL_V2, pJ246/STEL_V3, pJ246/STEL_V4, pJ246/STEL_V5
harboring the
gene encoding the tropoelastin 1-5 sequences, respectively were
optimized by DNA
2.0 (Menlo Park, CA) and they were employed as sources of the gene.
These five
sequences encode the same amino acid sequences of tropoelastin
while having
different GC content and were expected to have different expression
levels. These five
genes were expressed in different vector systems to determine the
optimal conditions
for high-level protein expression using regular induction. To
enhance the expression
yield, auto-induction was used for later experiments in different
cell lines.
Polyhistidine tagged tropoelastins at N-terminal were constructed
and expected to be
more stable than the native protein. Future studies with the
purified tropoelastin may
include cross-linking studies and incorporation of proline
analogues to investigate the
structure of tropoelastin.
Table of Contents
Table of contents
Introduction..............................................................................................................1
Materials..................................................................................................................9
Experimental
Methods................................................................................................12
Results and
Discussion...............................................................................................20
Conclusions
............................................................................................................35
References..............................................................................................................36
List of Figures
Figure 1. Structure of human
tropoelastin................................................................
..3
Figure 2. Process of
Elastogenesis.............................................................................4
Figure 3. Plasmid map of pYP34-38 based on pQE
80L..................................................13
Figure 4. Plasmid of map of pYP44-48 based on pBAD
system........................................14
Figure 5. 1% agarose gel electrophoresis analysis for
recombinant plasmids by
double
digestion.....................................................................................................21
Figure 6. Control elements of the pET
system.............................................................22
Figure 7. SDS-PAGE analysis of tropoelastin expression in
pBAD/HisA vector.....................23
Figure 8. Proposed structure for the C-terminal domain of
tropoelastin............................24
Figure 9. SDS-PAGE & western blot analysis of
auto-induction expression
results in
BL21.......................................................................................................26
Figure 10. Western blot analysis of whole cell lysate of
pYP64-pYP68 in
BLR(DE3)..............................................................................................................27
Figure 11. SDS-PAGE analysis with silver stain of pYP64
expression samples
from regular expression method in E. coli strain BLR
(DE3)..............................................28
Figure 12. SDS-PAGE analysis with silver stain of pYP65
expression samples
from regular expression method in E. coli strain BLR
(DE3)..............................................29
Figure 13. SDS-PAGE analysis with silver stain of pYP66
expression samples
from regular expression method in E. coli strain BLR
(DE3)..............................................30
Figure 14. SDS-PAGE analysis with silver stain of pYP67
expression samples
from regular expression method in E. coli strain BLR
(DE3)..............................................31
Figure 15. SDS-PAGE analysis with silver stain of pYP68
expression samples
from regular expression method in E. coli strain BLR
(DE3)..............................................32
Figure 16. SDS-PAGE analysis with silver stain of purified
protein samples
from regular expression method in E. coli strain BLR
(DE3)
after
dialysis.........................................................................................................33
List of Tables
Table 1. Diseases of elastin
fibers....................................................................................2
Table 2. DNA sequences for polyhistidine tag
oligonucleotide................................................15
About this Master's Thesis
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