1. TEMPLATE DESIGN © 2008
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Inserting Wild Type Calreticulin into the pRSET-A Vector
Ashia S Williams, Lystranne Maynard PhD, Akintunde Akinyemi MS.
Department of Chemistry, Howard University, Washington, DC 20001
Goal
The Purpose
In this study, ampicilin resistant mCRT was cloned in
order to understand the acetylation mechanism of
CRT outside the ER. To achieve this, wild type CRT
DNA was removed from a pGEM T-Easy vector and
placed into a the more useful pRSET-A vector.
The Significance
Calreticulin (CRT) is a conserved multifaceted protein
localized in the endoplasmic reticulum (ER) whose
roles inside the ER include calcium regulation and
protein folding inside cells. It has been shown that
acetylation, a post-translational modification, is the
mechanism by which CRT fulfills some of these roles
within the ER. Previous studies have localized CRT in
the endoplasmic reticulum however, recent studies
have found traces of this protein in liver, brain and
other cellular regions.
Background Information
Methods
Instrumentation
•Centrifuge
–DNA mini prep
•260nm UV/Vis Absorption of DNA
–260nm absorption of DNA
–260/280 is used to check for purity
–mCRT 99.1ng/uL
–pRSET-A 83.2ng/uL
•Incubator and Incubator Shaker
–Transformation and Overnights
•37 degree water bath
–digestion
Developing Protocol
Initially, the calreticulin DNA was cloned out of a
pGEX vector and attempted to be inserted directed
into pRSET-A vector using Ncol and BamH1 enzymes
in a simultaneous digestion. However, due to steric
hindrance, mCRT was unable to be inserted into the
vector.
Next, HindIII and BamH1 primers were used to
introduce these endonuclease sites to the ends of the
mCRT in pGEX and then insert the mCRT into a T-
vector in a sequential digestion. This step was
successfully completed.
It was concluded that inserting mCRT into a T-vector
would make it easier to extract the mCRT from the T-
vector and then insert it into a pRSET-A vector.
.
Results
Gel
pRSET-A
Small is the mCRT
Large T-vector
Excise
160mg mCRT
100mg pRSET-A
Medical Application Human Health and Disease
Conclusion
Acknowledgement
Absence of CRT is embryonically lethal
Presence of non-ER extracellular CRT
Rheumatoid Arthritis
Inflammatory bowel disease
Alzheimer Disease
Upon insertion of mCRT into the pRSET-A vector a
resistance to ampicilin is inherited therefore a
successful insertion will be indicated by the growth of
colonies in a LB-AMP environment.
Further Investigation
The resulting DNA can be further confirmed via DNA
sequencing. To develop a computational model of a
novel receptor, a controlled study will be performed on
wild type and mutant CRT to examine the role
acetylation may play in the exit of CRT from the ER
Discovery of Calreticulin (CRT)
•1974 Skeletal Muscle cells
•Thomas Ostwald and David MacLennan
•Conserved protein that assist in protein chaperoning
and calcium regulation in the endoplasmic reticulum
(ER).
–A network of membranous tubules within the
cytoplasm of a eukaryotic cell
•Other names
–HABP, Calregulin
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Structure
pRSET-A
T-vector
mCRT
DNA ladder
Lystranne Maynard, PhD
Akintunde Akinyemi, MS
Ronald E. McNair Program
Howard University-Funding
Department of Biology
Department of Chemistry
Winston Anderson PhD, Biology