Cloning and Expression of a Haloacid Dehalogenase Enzyme By: Skyler - - PowerPoint PPT Presentation

Cloning and Expression of a Haloacid Dehalogenase Enzyme

By: Skyler Van Senior Research Advisor: Dr. Anne Roberts

Outline

• The gene being cloned is JHP1130 from Helicobacter pylori (H.

pylori)

• JHP1130 is a member of the Haloacid Dehalogenase (HAD)

superfamily

• The gene was cloned into a pet21b vector for subsequent

expression

• The plasmid was transformed into BL21 E. coli cells for protein

expression

• The expected molecular weight of the desired protein was

roughly 25 kDa and a band at this molecular weight confirmed the presence of the correct protein.

• H. Pylori bacterium

E-ASP H Cl COO- R H OH COO- R E-ASP intermediate Cl- E-ASP

E-ASP P O OR O-

• O

E-ASP ROH E-ASP intermediate P O OH O-

• O

E-ASP intermediate E-ASP Mannose-6-Phosphate E-ASP Mannose-1-Phosphate

Dehalogenases Phosphatases Phosphomutases P-type ATPases (Ion pumps)

N N N N NH2 O H OH H H H H O P O- O O P O- O O P

• O

O- O E-ASP ADP E-ASP intermediate E-ASP + Pi

E-ASP intermediate E-ASP Mannose-6-Phosphate E-ASP Mannose-1-Phosphate

E-ASP P O OR O-

• O

E-ASP ROH E-ASP intermediate P O OH O-

• O

E-ASP H Cl COO- R H OH COO- R E-ASP intermediate Cl- E-ASP

Members of the Haloacid dehalogenase (HAD) superfamily have diverse functions

JHP 1130

• Putative member of the HAD phosphatase superfamily, members of

which are found in many metabolic pathways

O O P R O O O P O O O

• O

O

R Pi

• O

O

H2O

E-ASP Phosphorylated Enzyme intermediate

MOTIF I DXDX[T/V][L/V]

(P-type ATPases) DKTGTL

first aspartate becomes phosphorylated second aspartate involved in binding Mg2+ required for activity

MOTIF II [S/T]XX

involved in phosphoryl oxygen binding

MOTIF III K-[G/S][D/S]XXX[D/N]

(HAD) SSXXXD

involved in phosphoryl oxygen binding and binding of Mg2+

Had members can be identified by the presence of specific sequence motifs

PCR of JHP1130

• Forward primer (Nde1):
• 5’ – GCT GAC CAT ATG GCG CTT GAA GTG GTT TTA TGG – 3’
• Reverse primer (Ecor1 w/o histag):
• 5’ – GCA GTC GAA TTC TTA CTC TTT TGC GAA GTT TTG TAA ATC – 3’
• Reverse primer (Xho1 w/ histag):
• 5’ – ACA TCG CTC GAG CTC TTT TGC GAA GTT TTG TAA ATC – 3’

 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 Requirements for PCR:

• dNTP’s
• genomic DNA
• primers
• polymerase
• Mg2+
• small amount
• f ddNTP

pET–21 b (+)

General cloning strategy

pET21b  Plasmid Cleaved pET21b  Plasmid JHP1130  gene insert Plasmid w/ insert  integrated

• -CA
• -GTAT

TATG AC-- Insert Plasmid + CA GTAT TATG AC DNA LIGASE

Cleaved Gene Insert and Plasmid

• Ran gels containing insert and plasmid cleaved with each of the

restriction endonucleases

• Cut out the portion of gel with the bands corresponding to each

product

• Extracted the DNA from the gel for each product.
• This ensured that the products were free of impurities before ligation

reaction was performed.  nde1/xho1 insert w/ histag  nde1/ecor1 insert w/o histag  nde1/ecor1 plasmid digest w/o histag  nde1/xho1 plasmid digest w/ histag

Transformation of JHP1130 plasmid into DH5α cells

Luria Bertani broth w/ ampicillin

DH5α cells :

• The plasmid with the insert incorporated is grown with

DH5α cells

• Specially designed to allow passage of plasmid through cell

membrane

• This permeability makes the cells very fragile

Ampicillin/agar plate

Testing for JHP1130 insert in Plasmid

 DNA ladder  #1 T7 promoter/terminator PCR reaction  #2 T7 promoter/terminator PCR reaction  Plasmid control

Plasmid containing JHP1130 transformed into BL21 cells

From left to right: Protein standard, BL21 Transformation #1, BL21 Transformation #2, w/ IPTG #1, w/ IPTG #2

Isopropyl β-D-1- thiogalactopyranoside(IPTG):

• IPTG is used for protein

expression

• It mimics allolactose, but not

as easily degraded

• Binds to lac repressor

allowing transcription (JHP1130)

75 kDa  50 kDa  25 kDa 

Purification via Ion Exchange

JHP1130 facts:

• pI = 5.67
• M.W. = 25440.9 Da
• Number of Amino Acids = 222

Ion exchange chromatography setup:

• pH =8 (protein is negatively charged)
• Q sepharose anion exchange column
• to elute protein increase the salt

concentration

75 kDa  50 kDa  25 kDa 

Increasing [NaCl]

Ammonium Sulfate Fractionation

From right to left: protein ladder, 20%, 40%, 55%, 65%, 80%, protein ladder Saturated Ammonium Sulfate facts:

• Different proteins will

precipitate at different percentages

• JHP1130 has a molecular

weight of ~25 kDa

• The first lane with a band at

~25 kDa is 55% saturation

75 kDa  50 kDa  25 kDa 

Future research

• Purify protein to homogeneity
• Begin testing various small molecule

phosphorylated substrates to narrow down in vivo substrate

Questions