Cloning and Expression of a

Haloacid Dehalogenase Enzyme

By: Skyler Van

Senior Research Advisor: Dr. Anne Roberts

Skyler Van

Senior Research

March 22, 2011

Cloning and Expression of a Haloacid Dehalogenase Enzyme

Introduction:

The gene that I cloned is from a bacterium known as Helicobacter Pylori (H. pylori).

This gram negative bacterium has been linked to the formation of ulcers in the stomach as well

as gastric cancer. H. pylori is able to survive the acidic environment of the stomach by inserting

itself between the mucosal layer of the stomach and the stomach lining.

According to the article titled “Helicobacter pylori environmental interactions: effect of

acidic conditions on H. pylori-induced gastric mucosal interleukin-8 production”:

Helicobacter pylori have also developed mechanisms that allow the bacteria to survive

and remain motile in an acidic milieu. After the motile organism is ingested, it migrates

through the gastric contents, penetrates through and beneath the mucus layer, and

attaches to the gastric mucosal surface. Attachment to the gastric mucosal surface results

in bacterial–host interaction leading to a marked inflammatory response with infiltration

of neutrophils, lymphocytes, monocytes and plasma cells (Choi).

This interaction with the mucosal layer may contribute to the formation of ulcers. However, the

fact that H. pylori needs to migrate inside the mucosal layer suggests that it can’t survive in the

acidic pH of the stomach for long. In order to prevent the pathogenicity of H. pylori, it is

necessary to understand and be able to inhibit the enzyme action of the bacteria. The enzyme that

I am focusing on is JHP1130. This enzyme may not be responsible for the pathogenicity of H.

pylori. However, understanding the various enzymes associated with this type of bacteria and the

substrates that bind to them could aid in the classification of other bacteria that have enzymes

sharing JHP1130’s unique pathway.

JHP1130 is a member of the Haloacid Dehalogenase (HAD) superfamily. There are

several types of enzymes associated with this superfamily, which include phosphtases,

epimerases, and dehalogenases. Each of these HAD enzymes share a common catalytic

mechanism, which is the utilization of an aspartic acid intermediate. JHP1130 is a putative

phosphatase. However, it lacks homology with other phosphatases present in other types of

commonly found bacteria. Members of the HAD superfamily can be identified by several motifs

unique to these enzymes (Lee). The method of action for these enzymes utilizes two aspartic acid

(Asp) residues. The first Asp residue acts through nucleophilic catalysis. The second Asp residue

acts through a general acid/ base catalysis. The second Asp residue protonates the oxygen of the

leaving group and deprotonates the water or alcohol nucleophile. The leaving group is the result

of the initial nucleophilic reaction from the first Asp residue. There is also a Lysine or Arginine

residue that helps to position the Asp nucleophile and two carboxylate groups. This results in

easier binding of the Mg2+ cofactor (Allen).

Research methods and results:

When beginning my research, the first thing I did was perform a Polymerase Chain

Reaction (PCR) on the genomic DNA insert JHP1130. In order to perform PCR, I needed several

things. I needed a DNA template for my insert, a forward primer (Nde1), a reverse primer (Ecor1

w/o histag and xho1 w/ histag), a polymerase, all dNTP’s, a small amount of ddNTP, and Mg2+.

In order to purify the amplified JHP1130 insert, I used the Sure Clean procedure to remove

impurities.

Purity was confirmed by running the PCR solution on a 0.8% agarose gel with ethidium

bromide mixed in with the agarose. Ethidium bromide is an intercalating agent that inserts itself

between the base pairs in DNA rigidifying it. When hit with ultraviolet light, ethidium bromide

fluoresces orange. This means that the greater the concentration of DNA is the sample being run,

the greater the intensity of light given off. Since the JHP1130 has amino acids base pairs, it will

have 666 base pairs. When compared to the DNA ladder, the PCR bands all fell between the 600

Insert w/ histag Insert w/ histag Insert w/ histag Insert w/ histag

Insert w/o histag Insert w/o histag Insert w/o histag Insert w/o histag DNA ladder

and 700 base pair bands. The next step was to cleave both the plasmid and the inserts, both with

and without the histag, with the same restriction endonuclease. Doing this ensured that the

plasmid and the insert had complementary ends. This complementarity allowed the ends of the

insert to connect to the ends of the plasmid like a zipper. However, at this step something went

wrong and the plasmid re-ligated before the insert could be incorporated into the plasmid. At this

point, I had to start over from the beginning.

My second time through, I followed the same steps as the first time. However, I

performed an extra purification step on both the plasmid and the insert. After cleaving both with

the same restriction endonuclease, I ran both on an agarose gel. I then sterilized a razor blade

with acetone and a flame and cutout the desired bands of plasmid and insert. This ensured that

only the desired product was in solution when attempting to incorporate the JHP1130 insert into

the plasmid. In order to ensure the plasmid didn’t re-ligate before incorporation of insert, I

dephosphorylated the ligase. Next, the insert containing plasmid was transformed into DH5α

cells. The DH5α cells are specially designed to be permeable to the insert containing plasmid.

The caveat to this permeability is that they are extremely fragile. The plasmid/DH5α cell mixture

couldn’t be pippetted up and down to mix the solution because the cells would be destroyed.

Mixing was accomplished by gently tapping the centrifuge tube. This solution was then grown in

some Luria Bertani (LB) broth with ampicillin in it. These transformed cells are resistant to

ampicillin and are able to grow in solution. Since bacteria in the air won’t be resistant to

ampicillin, you can ensure that the only thing growing in the LB broth will be the transformed

cells containing the JHP1130 plasmid. After a couple hours in the shaking incubator, the solution

is pippetted onto an agar plate. The plate was left in the incubator over night to grow colonies of

DH5α cells transformed into JHP1130 insert containing plasmid.

I verified that the insert was in the plasmid by running another agarose gel. If the insert

had not been incorporated, then the bands corresponding to the T7 promoter/terminator PCR

reactions would be at the 200 base pair mark. The gel showed those bands to be at the 800 base

pair mark, which is consistent with insert incorporation.

The next step was to transform the insert containing plasmid into BL21 cells for protein

expression. Isopropyl β-D-1-thiogalactopyranoside (IPTG) is necessary for protein expression.

IPTG is similar to allolactose, which is needed to activate the lac operon. The difference between

IPTG an allolactose is that IPTG can’t be broken down by the enzymes used to degrade

allolactose. As a result, the lac operon will always be on and will continuously express the

JHP1130 protein. The lac operon is capable of expressing the JHP1130 protein because the

cleavage site that was used to insert the JHP1130 insert was in close proximity to the lac operon.

Once expressed, the protein is obtained by sonciating the transformed BL21 cells to break them

DNA ladder

#1 T7 promoter/terminator PCR reaction

#2 T7 promoter/terminator PCR reaction Plasmid control

open and release the protein inside. In order to separate the protein from the lysed cells, the

mixture is then centrifuged. The supernatant is drained off and saved for purification.

The band at 25 kDa shows that JHP1130 protein was present in the supernatant. The darkness of

the band indicates that the concentration of the protein was relatively high.

The two types of purification that I tried were anion exchange chromatography and

ammonium sulfate fractionation. Anion exchange chromatography uses a positively charged

stationary phase to attract negatively charged product. In order to elute the product, an increasing

concentration of sodium chloride will be run through the column and the flowthrough will be

collected. The JHP1130 protein has an isoelectric point of 5.67. This means that there are more

acidic amino acid residues in the protein than basic amino acids. The protein containing solution

was kept at a pH of 8. Aspartic acid and glutamic acid will be deprotonated at pH 3.35 and pH

4.25. Cystine isn’t deprotonated until pH 8.18, but some cystine residues may be deprotonated at

this pH, which will contribute to the overall negative charge. At pH 8, JHP1130 will have an

overall negative charge. This property was exploited for anion chromatography.

75 kDa 50 kDa

25 kDa

Increasing [NaCl]

This protein gel shows that the JHP1130 protein eluted quickly at low concentrations of sodium

chloride. This mean that for some unknown reason the protein didn’t stick to the column even

though having an overall negative charge it should have stuck to the positively charged column.

One possibility is that there was some other negatively charged molecule in solution. If the

negatively charged molecule or molecules were more negatively charged, then they could have

preferentially bound to the column over the desired protein. The reason for this is that the more

negatively charged a molecule is the greater the attraction between the molecule and the

positively charged stationary phase. The results from my second purification method as well as

the other bands at various other molecular weights present on the protein gel above could support

that theory.

The second method of protein purification was using a saturated ammonium sulfate

solution to precipitate out impurities. The basis for this method of protein purification is that

75 kDa 50 kDa

25 kDa

different proteins will precipitate out of solution at different concentrations of saturated

ammonium sulfate. Increasing [Saturated Ammonium Sulfate]

This gel shows a band at 25 kDa, which illustrates that the JHP1130 protein starts to precipitate

out of solution at a 55% saturated ammonium sulfate concentration. No bands seem to be visible

at a 20% concentration, which suggests that no proteins precipitate at that concentration. At a

40% concentration there were several higher weight proteins that precipitated out of solution, but

there was no JHP1130 protein because there was no band at the 25 kDa mark. Since a band was

present at 25 kDa for a 55% concentration and no band at the same weight for a 40%

concentration, it is safe to assume that a concentration between these percentages will precipitate

out the maximum amount of undesired proteins while leaving the JHP1130 protein in solution. In

order to purify the protein solution, a 50% saturated ammonium sulfate concentration was used.

75 kDa 50 kDa

25 kDa

Future research:

If the reason that the JHP1130 protein didn’t stick to the anion exchange column was that

a more negatively charged protein preferentially bound to the column in its place, then

precipitating some of the other proteins in solution with the ammonium sulfate method could

have solved that problem. Hopefully, without those impurities the JHP1130 protein will stick to

the column and can be furthered purified. Another possibility for purification is size exclusion

chromatography. After ammonimum sulfate fractionation, the JHP1130 protein should be one of

the highest weight protein left in solution. Size exclusion chromatography works by using small

porous beads to differentiate between higher larger molecules and smaller molecules. A larger

molecule will elute first because it passes through the column unimpeded. Smaller molecules can

get caught in the pores of the beads and will take longer to elute. Since the desired protein should

be the highest molecular weight molecule in solution, it will come out faster than the rest of the

proteins in solution.

After those steps further purification may be required. Once the solution has been

purified to homogeneity, testing of the enzyme can begin. Testing will consist of using various

small molecule phosphorylated substrates to narrow down the in vivo substrate. Since JHP1130

is a phosphatase with a unique metabolic pathway, finding the substrate compatible with the

protein could be useful in the future. As of now, other bacteria’s phosphatases don’t share the

same pathway as JHP1130. However, that doesn’t mean that there aren’t other bacteria that have

the same or a similar metabolic pathway.

Works Cited

Choi, Il Ju, Saori Fuhimoto, David Y. Graham, Yoshio Yamaoka, Kazuyoshi Yamauchi.

“Helicobacter pylori environmentl interactions: effect of acidic conditions on H. pylori

induced gastric mucosal interleukin-8 production.” Cell Microbiol. 9 Oct. 2007: 2457-

2469

Lee, Seok-Yong, Emma McCullagh, Anne Roberts, Ruth E. Silversmith, David E. Wemmer.

“YbiV From Escherichia coli K12 is a HAD Phosphatase.” Proteins: Structure, Function,

and Bioinformatics. 2004

Allen, Karen N., Debra Dunaway-Mariano, Sushmita D. Lahiri, Guofeng Zhang.

“Diversification of Function in the Haloacid Dehalogenase Enzyme Superfamily: The

Role of the Cap Domain in Hydrolytic Phosphorus-Carbon Bond Cleavage.” Bioorg

Chem. 27 Oct. 2006: 394-409