the effect of thioredoxin on the solubility of proteinase inhibitor 2 in an escherichia coli...

THE EFFECT OF THIOREDOXIN ON THE SOLUBILITY OF PROTEINASE INHIBITOR 2 IN AN ESCHERICHIA COLI OVEREXPRESSION SYSTEM.

1-beta – David, Chris, Ivan, Josef

OVERVIEW Introduction

• Background information• Hypothesis• Experimental aims

Methodology• Procedures• Results

Conclusion• Summary of conclusions• Future directions• Acknowledgements

INTRODUCTION Background Information

• Overexpression of proteins in bacteria is a fast and cheap way to generate large quantities of protein.

• Overexpressed proteins often form insoluble protein aggregates known as inclusion bodies.

• Fusion of genes with a thioredoxin gene increases solubility of proteins and reduces the amount of inclusion bodies.

Experimental Question• What about in trans? Could thioredoxin be

simultaneously expressed with the protein of interest to enhance its solubility?

Hypothesis• Overexpression of PI2 along with the

expression of thioredoxin will lower the amount of inclusion bodies.

• PI2 = proteinase inhibitor 2 (our protein of interest)

• Proposed mechanism: Reduction of thiol residues.

Experimental Aims

• Transform different plasmid constructs containing PI2 and thioredoxin into BL21 (DE3).

• Induce protein overexpression.

• Measure amount of inclusion bodies.

• Compare the amount of inclusion bodies between different thioredoxin constructs (in cis or trans).

METHODOLOGY Plasmid Constructs

• pET32a/PI2+PI2 gene with a fused thioredoxin gene.

• pET32a/PI2+∆trxPI2 gene with a deleted thioredoxin gene.

• pET32a/PI2+∆trx/trx+PI2 gene with a non-fused thioredoxin gene.

• pET32aGeneric construct only the thioredoxin gene.

JP61/62

JP63/64

CD111-114

Transformation Made competent BL21 (DE3) cells using CaCl2. Heat-shock transformation. Successful transformation of JP61-64 and CD111-

114 into our overexpression system.

Confirming Plasmid Constructs – RE Digestion

FIG 1. Restriction digestion of JP and CD plasmid constructs.

No bandsat 590 bp!

Confirming Plasmid Constructs – Sequencing

• pET32a/PI2+PI2 gene with a fused thioredoxin gene.

• pET32a/PI2+∆trxPI2 gene with a deleted thioredoxin gene.

• pET32a/PI2+∆trx/trx+PI2 gene with a non-fused thioredoxin gene.

• No sequencing was done in previous studies (J. Park (2008) and C. Duronio (2012)).

Missing PI2!

Missing PI2!

Missing PI2!

Change in Game Plan

• We do not have any functional PI2 constructs to work with.

• We do have DH5α containing PE32PI2.

• Pe32PI2 is the original plasmid containing PI2.

• Performed plasmid purification on PE32PI2.

• Used PCR to amplify the PI2 sequence.

PCR Need to obtain enough PI2 to make new constructs for our

experiment and for sequencing. Performed PCR using varying volumes of DMSO (2.5, 1.5,

0.5, 0 microlitres).

Lane 1: 100bp ladderLane 2: HindIII ladderLane 3: Positive controlLane 4: Negative controlLane 5: PCR w/ 2.5 ul DMSOLane 6: PCR w/ 1.5 ul DMSOLane 7: PCR w/ 0.5 ul DMSOLane 8: PCR w/ 0.0 ul DMSO

FIG 2. 1.0% agarose gel electrophoresis of PCR products for 1h at 125V.

Got a product!

Gel Extraction and More PCR Performed a gel extraction on the band from the previous

gel. Used PCR to amplify the product.

Lane 1: 100bp ladderLane 2: HindIII ladderLane 3: Positive controlLane 4: Negative controlLane 5: PCR w/ 2.5 ul DMSOLane 6: PCR w/ 1.5 ul DMSOLane 7: PCR w/ 0.5 ul DMSOLane 8: PCR w/ 0.0 ul DMSO

FIG 3. 1.0% agarose gel electrophoresis of PCR products for 1h at 125V.

No products.

CONCLUSION Summary

JP61-64 designed by Jei Park (2006) did not contain PI2.

Since CD111-114 designed by Chris Duronio (2012) was adapted from JP61-64, they also did not contain PI2.

PE32PI2 confirmed to have PI2 by sequencing.

Direction of the project has been changed to construct proper PI2 constructs.

Future Directions

• Make the PI2/thioredoxin constructs properly.

• Transform each plasmid into BL21 (DE3) E. coli.

• Induce protein expression.

• Look for inclusion bodies.

ACKNOWLEDGEMENTS We would like to thank:

• Dr. Oliver

• Chris Deeg (TA)

Special thanks to:• Other groups

• Wesbrook Media room

• UBC for funding the project