This document summarizes research on expressing and purifying serine collagenases from the Aedes aegypti mosquito in order to better understand their roles in blood meal digestion. Key findings include:
- AeSPV was successfully expressed in E. coli but not in soluble form, requiring refolding techniques for purification.
- AaLT was expressed both as a zymogen (AaLT-Z) and with an enterokinase cleavage site (AaLT-Mek) to activate it. Both forms were activated by different mosquito proteases.
- Future work will test the protease activities against specific substrates and elucidate their roles in blood meal digestion. This research could inform new mosquito-
Oligosaccharide Analysis Using High-Performance Anion-Exchange Chromatography...
2015 UCSC ACS Talk
1. Recombinant Expression and
Purification of Serine Collagenases
from the Ae. aegypti Mosquito
Bharat Patel and Taylor McCann
Department of Chemistry
San Jose State University
May 9th, 2015
UCSC
ACS 27th Annual Undergraduate Research Symposium
3. Transmission
• Imbibes from Viremic Host
• Transmits to Uninfected Host
• In Aug-Nov 2009, 22 cases of
Dengue Fever reported that were
locally acquired
• Chikungunya= 2,492 cases (11 of
which were locally acquired) as of
Feb 2015
Aedes aegypti
Dengue
Flavivirus
Yellow Fever
Flavivirus
Chikungunya
Alphavirus
4. Current Strategies for Control
• Insecticides
• Pharmacological drugs
• Transgenic mosquitos Mosquito-Selective
Inhibitors
Blood Metabolism
5. Ae. aegypti blood meal digestion
• Hundreds of serine
protease-like genes in
Ae. aegypti genome
BUT only a few, highly
abundant proteases in
midgut
• Only expressed after blood
meal
• Serine Proteases from
mosquito
• Trypsin-, chymotrypsin-,
serine collagenase-like
Blood Meal (Low Complexity)
80% Total Protein:
Hemoglobin (~330 μg)
Serum Albumin (~50 μg)
Immunoglobulin (~15 μg)
2 mg female mosquito
>2 μl blood
Digestion ~30-40 hrs
6. What is a protease?
An enzyme that catalyzes
cleavage of peptide bonds at
specific amino acids
A serine collagenase breaks
peptides in collagen
Main proteases known to be
involved in Aedes aegypti blood
meal digestion so far:
AaET
AaLT
AaSPVI
AaSPVII
Why do mosquitos need an
enzyme that breaks down
collagen if there is no
collagen in blood?
7. AaSPV
Expressed at constant levels in the midgut during blood
meal acquisition
Its catalytic triad has been altered slightly
What is it’s role?
Asp-His-Ser Asp-Leu-Ser
9. Results
Growth conditions:
T7 shuffle cells
LB media
15 C
0.1mM IPTG
Samples taken at 0, 1, 2,
4, 22, 24, and 26 hours
Total expression is good
But no soluble
expression
MW of AaSPV without
leader sequence= 27.5
kD
10. Refolding Scheme on Column
1. Insoluble protein unfolded
using 6M GuHCl
2. Loaded to Nickel column
3. Decreasing concentrations
of GuHCl to refold on
column
4. Thorough wash with 0M
GuHCl
5. Increasing concentrations
of Imidazole to elute
16. Soluble AaLT-Mek Activation by rEK
MW 0 5 10 15 30 45 60 90 120 180 (min)
Simply Blue Stain
30
25
-AaLT-Mek soluble
expression
-Purified using Ni2+
column
-Treated with rEK
-Incubated at 4oC (in
ice)
-Samples taken at
selected time points
N-terminally His6-
tagged AaLT-Mek:
~27.6kDa
“Active” AaLT-M:
~24.2 kDA
17. Soluble AaLT-ZCleavage by AaSPVI
-AaLT-Z soluble
expression
-Purified using Ni2+
column
-Treated with AaSPVI
crude lysate
-Incubated at 4oC (in ice)
-Samples taken at
selected time points
N-terminally His6-
tagged AaLT-Z:
~27.6 kDa
“Active” AaLT-M:
~25.7 kDA
MW L 0 0.5 1 2 3 4 5 6 8 10 (min)
Simply Blue Stain
30
25
18. Soluble AaLT-ZActivation by AaSPVII
Simply Blue Stain
30
25
-AaLT-Z soluble
expression
-Purified using Ni2+
column
-Treated with AaSPVII
crude lysate
-Incubated at 4oC (in
ice)
-Samples taken at
selected time points
N-terminally His6-
tagged AaLT-Z:
~27.6kDa
3 different bands?!
MW L 0 0.5 1 2 3 4 5 6 8 10 (min)
19. Soluble AaLT-ZCleavage by AaET
Simply Blue Stain
30
25
-AaLT-Z soluble
expression
-Purified using Ni2+
column
-Treated with AaET
crude lysate
-Incubated at 4oC (in
ice)
-Samples taken at
selected time points
N-terminally His6-
tagged AaLT-Z:
~27.6kDa
“Active” AaLT-M:
~25.7 kDA
MW L 0 0.5 1 2 3 4 5 6 8 10 (min)
20. Future Studies
• To determine the role of serine collagenases in blood
meal digestion
• To test specific substrates against our proteases and see
activity
21. Acknowledgements
AaET
Jamie Gallimore
Anh-Dai Nguyen
AaSPVI
Radhakrishna Patel
Eliza Vien
Tejpal Kang
AaSPVII
James Nguyen
AaLT
Jonathan Fong
Rascón Lab Members
Jennifer Le (JHA15)
Alexia Perryman
(AaSPV)
Justin Tran
(AaSPII/AaSPIV)
Joshua Garcia (AaSPI)
Mai Lee (AaSPV)
Olive Burata
(AaCHYMO)
Rachael Lucero
(AaCHYMO)
Diane Eilerts - MS
(AaSPIV)
Kamille Parungao
Jacob Hickey
Canaan Belete
Marina Dragovic
Dr. Alberto A. Rascón, Jr.
THANK
YOU!
The female Aedes aegypti mosquito must acquire a blood meal from a human host in order to obtain the proper nutrients required for completion of the gonotrophic cycle (so this gonotrophic cycle is the process of blood-feeding, egg maturation, and oviposition (or egg laying). Once she imbibes or drinks a blood meal, the release of midgut proteases are induced and released, which leads to the digestion of the blood meal proteins and the release of the required nutrients. After metabolizing the meal, the mosquito then lays her eggs near the surface of standing water, where she can lay between 100-200 eggs. The eggs can take between 2-7 days to hatch, but can survive up to 3 months or more without hatching in a stage called diapause. Once they hatch, they hatch into larvae, followed by a pupation stage, and after about 1-4 days they become adult mosquitoes. Once an adult, they immediately seek a mate and a blood meal, and the whole process can begin all over again.
Now, this blood feeding behavior has facilitated the spread of blood-borne pathogens from the mosquito to humans. The Ae. aegypti mosquito is an efficient biological vector of the Dengue fever (flavivirus), Yellow fever (flavivirus) and Chikungunya (Alphavirus) viruses. The mosquito becomes infected when it takes a blood meal from a viremic host and once infected it can transmit the virus to the an uninfected human host. In the mosquito, the virus infects and replicates in the midgut cells, which then travel to the salivary glands. So, when she takes a blood meal, the virus is transferred from the salivary glands of the mosquito to the human host. It has been shown that the Ae. aegypti mosquito can take more than one blood meal before its first gonotrophic cycle, so you can see how this contributes to its role as an efficient biological vector of these human pathogens.
HUMAN HOST (NOT ACTIVE)
Not “signal sequence” they cleave after specific sites (i.e: trypsins prefer to cleave….)
PCR to isolate SPV, clone into vector, plasmids inserted into T7 cells (know why important), growth experiments followed by SDS-PAGE to determine expression
Surprising because t7 cells should help with oxidizing cytoplasm which will lead to better soluble expression
http://2009.igem.org/wiki/images/thumb/d/dd/OldPurificationGraphic.png/500px-OldPurificationGraphic.png
We unfolded the protein using GuHCl, loaded the protein onto the column which the exposed His-tag should chelate to the column, then we refolded using decreasing concentrations of GuHCl follwed by increasing concentrations of imidazole to elute the protein off the column
FUTURe studies: we can repeat this again and repeat the purifcation steps again
-Not autocatalytic like the previous three proteases (AaET, AaSPVI, and AaSPVII).
-Was able to solubily express and purify the inactive form using a Ni2+ column.
-Upon amino acid sequence analysis and modeling, the closest results similar was with the heel fly serine collogenase at 40%), which was weird because if you recall to biology, there is no collagen in the blood.
Descirbe the gel: initial one pass Ni2+ purification (talk about the concentration from lower to the higher)
Express soluble
Homology modeling: takes the AA and compares the models with other hits. We expected the a trypsin like model
We were able to solubally express this but back then,
Graduate school: wasn’t able to express it solubally. So this allowed them to activate in vitro using the enterokinase construct (expressed insoluble and then was refolded using a similar scheme described by taylor and then activate it by EK)
To determine this, we need to isolate and activate the protease. And since we don’t know what the activator is, we have turned to the soluble expression of the AaLT-Mek
He was able to activate the enzyme and activity assays with all the proteases. Did not work with LT and baPNA under all of the conditions. Bapna is a trypsin substrate
To determine this, we need to isolate and activate the protease. And since we don’t know what the activator is, we have turned to the soluble expression of the AaLT-Mek
We treated mEK construct with rEK to see if we see a shift from the inactive to active form, but we are still trying to figure the correct substrates
1. COLLAGEN is OBVI!
They are expressed during the same time points so we are hypothesizing that one of these (SPVI or SPVII) is the activato for the LT)
In the beginning is the AaLT-Z but at the end of the gel it is “active”
AaSPVII
? kDA
In this case, same conditions, SPVII could just be digesting the LT
Soluble AaLT-Mek Activation- We also tested the
??? kDA
To our surprise, we saw that AaET was activating the LT like SPVI, and that weird because it is part of the early phase digestion.