Biomaterial Design: Human Tropoelastin Open Access

Pei, Yunyun (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/zc77sq52g?locale=en
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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

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