Megan Lasseter

Journal of Undergraduate Research
Volume 4, Issue 2 - October 2002

Construction and Expression of a Chimeric Protease in Hopes of Aiding the Refolding and Activation of the Mature Plasmepsin VI Enzyme

ABSTRACT

The present study attempted to produce a chimeric gene consisting of the prosegment of human pepsin and the complete gene of Plasmepsin VI, a malarial protein. The gene was created using overlap PCR, cloned, and expression was induced to produce a large quantity of the desired protein. Attempts to purify and kinetically assess the product were only moderately successful. Future projects will focus on these goals using our newly engineered DNA.

INTRODUCTION

Malaria is one of the most deadly diseases mankind has faced. Each year an estimated 300-500 million cases will result in 1-2 million deaths (1). Recently, there has been evidence to a worsening situation. Problems such as poor health care systems and increasing drug resistance have led to the need for new antimalarial agents. Current therapeutics include quinone based drugs which prevent the polymerization of heme released from the hemoglobin molecule during digestion. This causes heme to build up to toxic levels, resulting in the death of the parasite (2). Resistance to these drugs is rising, especially in Plasmodium falciparum, the strain which causes the most deadly form of the disease.

Due to the recent genome-sequencing project of Plasmodium falciparum, we now know that this parasite contains at least ten aspartic protease genes (3). Four of these genes code for the proteins that are responsible for hemoglobin degradation, the plasmepsins. These proteins, known as aspartic proteases, are bilobal enzymes that contain two aspartic acids in the active site cleft. These residues activate a water molecule, which acts as a nucleophile to cleave the scissile bond in the hemoglobin (4). It is crucial to express and kinetically characterize these enzymes in order to understand the specificity of substrate and inhibitor binding. Understanding subsite specificity will aid in the development of drugs designed to combat malaria.

One of the newly discovered aspartic proteases from the P.falciparum genome project, plasmepsin VI (PM6), is being looked at as a new drug target. Expression of the enzyme has not been successful thus far. Prosegment chimeras may help us to promote activation or aid in the expression and refolding of the protein. Classic aspartic proteases, like pepsin, have prosegments 44-49 amino acids in length. The plasmepsin prosegments are approximately 125 amino acids long (5,6). Past research has found that shortening the prosegment of plasmepsin II (PM2) to 49 amino acids allows for expression and refolding. Therefore, a chimeric gene consisting of the prosegment of human pepsin and the gene of plasmepsin VI will be created in hopes of achieving similar results. This chimera will be used to express the protein for the plasmepsin VI gene. Once the enzyme is refolded properly the human pepsin prosegment will be removed from the zymogen form, leaving only the native PM6 enzyme for study. The next step would involve purifying and kinetically analyzing the PM6 enzyme.

MATERIALS AND METHODS

Polymerase Chain Reaction (PCR) was performed on prosegment and the gene using specific primers synthesized by life technologies. Taq polymerase from Qiagen or Vent from New England Biolabs (NEB) was utilized to amplify the gene product in the presence and absence of Magnesium Sulfate from NEB. Both the amplification and overlap PCR products were electrophoresed on a 1.0% high melt agarose gel, and gel purified using a Qiagen Gel Purification Kit. Cloning of the amplification products into a pCR2.1Topo vector and TOP10F' cells were performed with a TOPO TA cloning kit from Invitrogen. Upon purification, the chimeric DNA was topo-cloned into a TOP10F' cell line. The plasmid DNA digested with BamH1 from New England Biolabs along with a pET3a expression vector from Invitrogen. All restriction enzymes, as well as T4 DNA ligase, were obtained from NEB. BL21(DE3)pLysS expression cells were purchased from Invitrogen. Fisher Scientific supplied all other reagents for buffers and media.Gel Filtration column (Superdex 75 media) was from Amersham Pharmacia.

RESULTS

Polymerase Chain Reaction (PCR) was utilized to create a chimera containing the prosegment of human pepsin and the gene portion of plasmepsin VI (PM6). When electrophoresed, bands were seen at approximately .5Kbs and 1Kbs. After the overlap PCR was performed the expected band was located at 1.2 Kbs. Post-dialysis was tested for kinetic activity and some active enzyme was shown to be degrading the standard substrate. Purification of this enzyme is the next step.

DISCUSSION

Overlap PCR

Four primers were designed for this experiment. Two of which were engineered to contain BamH1 restriction sites so that the chimeric gene could be easily cloned into an expression vector. The other primers contained complementary regions to both stands allowing for a genetic "overhang" to be created (Figure 1).

Figure 1. Experiment Primers.

The prosegment of human pepsin and the gene of PM6 were amplified in separate reactions using the aforementioned primers. The resulting DNA was then combined in another PCR reaction, which allowed the complementary regions to align. The addition of DNA polymerase to the reaction allowed the overhangs to extend, which created the chimeric gene. Lastly, the unique primers containing the BamH1 restriction site were added to the reaction to generate many copies of our chimeric gene.

Cloning Reactions

Both the amplification and overlap PCR products were electrophoresed on a 1.0% agarose gel, excised, and gel purified. Upon purification the chimeric DNA was topo-cloned into a TOP10F' cell line. The plasmid DNA was isolated and digested with Xba1 to verify insertion of our PCR product into the cloning vector. Positive clones were identified based on fragment size and these clones were sent for sequencing. Upon confirmation for a positive clone, it was digested with BamH1 along with a pET3a expression vector. These DNA fragments were utilized in a ligation reaction and were then transformed into a BL21(DE3)pLysS expression cell line.

Expression of Chimeric Gene

A positive clone verified by DNA sequencing was grown overnight in LB media with 1mM ampicillin (final concentration). Four liters of LB-Amp media were inoculated with 250mL of the overnight culture. When an OD₆₀₀ between 0.4-0.6 was observed, IPTG was added at a final concentration of 0.4mM to the flasks and 2mLs were collected. This was time zero. Every hour another two milliliters were collected, an OD₆₀₀ taken, and the rest of the sample was saved to be electrophoresed on a SDS-Page gel. After three hours the cells were harvested through centrifugation and lysed with a French Pressure Cell. Inclusion body (IB) purification was performed using centrifugation and resuspension over a 17% sucrose solution.

Refolding

The resulting IBs were resusupended at 50 mg/mL Tris-EDTA buffer and stored in 1mL aliquots at &endash;20ºC. The IBs were denatured using 8M urea and placed in 6-8kDa MWCO dialysis tubing. The PI3 protocol utilized two changes at pH11.0 and the remaining at pH 8.0.

The post-dialysis product was then applied to a Superdex 75 Gel Filtration column. Ideally, our properly folded protein would be delayed in the pores of the column and be collected after the improperly folded protein. Fractions were then collected in 1.5mL samples at a rate of 1.0mL/min. The graph of the fraction collection showed two distinct peaks in which protein was collected. However, there were still many impurities that showed up in the SDS-Page gel. The next step in this research will involve loading the post-dialysis on an ion-exchange column and attempting to collect a more purified product to use in kinetic analysis of the mature protein.

Future Projects

Malaria is continuing to claim the lives of millions of people every year. The ability of this disease to resist current therapeutics is on the rise. This knowledge makes finding new drug therapy vital. Aspartic proteases are excellent targets for these drugs and research on the function of these proteins is essential to finding their weaknesses. Utilizing chimeric proteins, such as prosegment chimeras, can aid in the expression and refolding of proteins that prove difficult to purify.

REFERENCES

Westling, J., P. Cipullo, S. Hung, H. Saft, J. Dame, and B. Dunn. Activity site specificity of plasmepsin II. Protein Science. 8:2001-2009, 1999.
Francis, S., D. Sullivan, Jr., and D. Goldberg. Hemoglobin metabolism in the Malaria parasite Plasmodium falciparum. Annual Review of Microbiology. 51:97-123, 1997.
Bowman, S., et al., The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum. Nature. 400:532-538, 1999.
Barreett, A. J., D. Rawlings, and J. Woessner. Handbook of Proteolytic Enzymes. Academic Press, Oxford. pp 828-836, 1998.
Davies, D. The structure and function of aspartic proteinases. Annual Review of Biophysics and Biophysical Chemistry. 19:189-215, 1990.
Foltmann, B., Structure and function of proparts in zymogens for aspartic proteinases. Biological Chemistry Hoppe Seyler, 369:311-314, 1998.

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