Jeong Keun Kim
304346888
Chem 153L
Lab Report 2
Induction and Analysis of His-tagged Fusion Protein Expression in E. coli
Introduction
The purpose of this lab was to produce and purify YqhD, alcohol dehydrogenase. To do
this, we placed E. coli in a culture that contained pETDuet, a plasmid that contains the yqhD
gene. The pETDuet plasmid also contains 6 Histidine residues. This hexa-histidine tag will later be
used to purify the target protein through affinity chromatography. After the plasmid has been
taken up by the bacteria, the over expression of the yqhD gene can take place. However, the
actual expression of the protein, yqhD, is regulated by Lacl-regulated promoters. To induce
expression, we added IPTG (isopropyl-thio-galacto-B-D-pyranoside) to induce the promoters and
onset the expression of the protein. We let the cells incubate for various different times then
place them into a protein assay. We assay for protein over-expression at different times to see
what size protein is being over expressed. For example, if we load a gel with a cell that has been
incubating for 1 hr with a cell that has been incubating for 3 hours; we can see which protein is
being over-expressed because the over-expressed protein will create a darker line in the cell that
was incubating longer.
In this lab we will be performing a coomassie stain SDS-PAGE and an immunoblot
(Western blot). The coomassie stain shows us that we successfully loaded proteins into the gel.
The immunoblot will show us our target protein; if the protein has been tagged successfully with
the 6 histidine residues.
Results
O.D.600nm
T0
.2456
T1
.4010
T1.5
.6157
The calculations for normalizing our samples (for loading the gels) are hand written, on a
separate sheet of paper.
During the Lab, the coomassie stained gel and the immunoblot did not show the expected
results, due to a faulty reagent. Because of this, I will be using the provided pictures of the
Coomassie stained gel and the immunoblot. In these 2 pictures, 4 time points were analyzed;
times 0, 1, 2, and 3 hours after induction were used.
Coomassie-stained SDS-PAGE
The gel above is the gel that my group obtained during the lab.
We did not get any results from our immunoblot
Discussion
The data obtained from the sample Coomassie stained SDS-PAGE and the immunoblot do
match the literature molecular weight of yqhD. According to EcoCyc the experimental molecular
weight of yqhD should be 43 kD. However, the molecular weight can also be 42.097 kD if the
weight was to be determined by the nucleotide sequence.
The results from both methods are consistent based on the sample gels that were
provided. By comparing the band that is getting gradually darker with the bands on the standard,
I was able to estimate that the band that was getting darker must have been between 35 and 50
for the unstained SDS-PAGE, and between 37 and 50 for the coomassie stained gel. These results
are consistent with the 43kD found in the literature.
Though the results from the sample gels were very close to the literature, there are some
places where error can occur. Error can occur due to the usage of SDS-PAGE. When SDS binds to
a protein it can change the shape of the protein, making the protein elute more or less. Also we
cannot be certain that our target protein is actually being expressed solely from the SDS-PAGE
because SDS-PAGE separates proteins out by size. There may be other proteins are the same size
as our target protein, which can lead to some error.
From this lab I learned that it is very important to load the same amount of cells per lane,
when we are trying to compare protein expression. When loading the gels, we made sure to
dilute our samples to make sure that we had the same amount of cells per lane. This is important
because, theoretically, the longer the cells have had time to incubate, the more protein
expression we should get. This can be hard to tell if there are uneven amounts of cells in each
lane. For example, if there are more cells in one lane, than that lane will have more protein
expression, and if there are less cells in another lane, that lane will show less protein expression;
not because of the difference in protein expression, but because of the number of cells. To make
sure we had the same amount of cells in each lane, we diluted each of our samples to a
normalized amount that was set at time 0.
To confirm that we had our protein of interest, we had to perform an immunoblot
(western blot). This is due SDS-PAGE only separating out protein by size. If there was a protein
that had a similar size as our target protein, then it will be hard to tell if we actually had our
protein. Immunoblotting takes care of this problem by using detection antibodies. They can
easily tell if our protein of interest is present by recognizing the tag that was placed on them.
Works Cited
EcoCyc. "Escherichia Coli K-12 Substr. MG1655 YqhD." Escherichia Coli K-12 Substr. MG1655
YqhD. EcoCyc: Nucleic Acids Research, 19 Oct. 2015. Web. 20 Oct. 2015.
http://biocyc.org/ecoli/new-image?object=G7564
"YqhD:Gene Product(s)." EcoliWiki. PortEco, 22 June 2015. Web. 20 Oct. 2015.
http://ecoliwiki.net/colipedia/index.php/yqhD:Gene_Product(s)