Cloning and Expression of a Haloacid Dehalogenase Enzyme. By: Skyler Van Senior Research Advisor: Dr. Anne Roberts

Transcription

1 Cloning and Expression of a Haloacid Dehalogenase Enzyme By: Skyler Van Senior Research Advisor: Dr. Anne Roberts

2 utline 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.

3 H. Pylori bacterium

4 Members of the Haloacid dehalogenase (HAD) superfamily have diverse functions Cl Cl - - H Cl intermediate R Cl intermediate C C P R P R Dehalogenases RH RH intermediate intermediate H H R H C C Phosphatases P H P H Mannose-6-Phosphate intermediate Mannose-1-Phosphate Mannose-6-Phosphate intermediate Mannose-1-Phosphate Phosphomutases - P - P - NH 2 N N N N ADP H H H H H H P intermediate - P-type ATPases (Ion pumps) + P i

5 JHP 1130 Putative member of the HAD phosphatase superfamily, members of which are found in many metabolic pathways R P - R P H 2 Pi - Phosphorylated Enzyme intermediate

6 Had members can be identified by the presence of specific sequence motifs MTIF I DXDX[T/V][L/V] (P-type ATPases) DKTGTL first aspartate becomes phosphorylated second aspartate involved in binding Mg 2+ required for activity MTIF II [S/T]XX involved in phosphoryl oxygen binding MTIF III K-[G/S][D/S]XXX[D/N] (HAD) SSXXXD involved in phosphoryl oxygen binding and binding of Mg 2+

7 PCR of JHP1130 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 Mg 2+ small amount of ddntp 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

8 pet 21 b (+)

9 General cloning strategy pet21b Plasmid Cleaved pet21b Plasmid JHP1130 gene insert Plasmid w/ insert integrated Plasmid --CA --GTAT + CATATG GTAT AC Insert TATG AC-- DNA LIGASE

10 Cleaved Gene Insert and Plasmid nde1/xho1 insert w/ histag nde1/ecor1 insert w/o histag nde1/ecor1 plasmid digest w/o histag nde1/xho1 plasmid digest w/ histag 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.

11 Transformation of JHP1130 plasmid into DH5α cells Luria Bertani broth w/ ampicillin Ampicillin/agar plate 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

12 Testing for JHP1130 insert in Plasmid DNA ladder #1 T7 promoter/terminator PCR reaction #2 T7 promoter/terminator PCR reaction Plasmid control

13 Plasmid containing JHP1130 transformed into BL21 cells 75 kda 50 kda 25 kda 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)

14 Purification via Ion Exchange JHP1130 facts: pi = 5.67 M.W. = Da Number of Amino Acids = 222 Increasing [NaCl] 75 kda 50 kda 25 kda Ion exchange chromatography setup: ph =8 (protein is negatively charged) Q sepharose anion exchange column to elute protein increase the salt concentration

15 Ammonium Sulfate Fractionation 75 kda 50 kda 25 kda 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 From right to left: protein ladder, 20%, 40%, 55%, 65%, 80%, protein ladder

16 Future research Purify protein to homogeneity Begin testing various small molecule phosphorylated substrates to narrow down in vivo substrate

17 Questions