publication . Master thesis . 2008

Construction of a system for heterologous production of carbonic anhydrase from Plasmodium falciparum in Pichia pastoris

Gullberg, Erik;
Open Access English
  • Published: 01 Jan 2008
  • Publisher: Linköpings universitet, Institutionen för fysik, kemi och biologi
  • Country: Sweden
Abstract
Malaria is one of the biggest current global health problems, and with the increasing occurance of drug resistant Plasmodium falciparum strains, there is an urgent need for new antimalarial drugs. Given the important role of carbonic anhydrase in Plasmodium falciparum (PfCA), it is a potential novel drug target. Heterologous expression of malaria proteins is problematic due to the unusual codon usage of the Plasmodium genome, so to overcome this problem a synthetic PfCA gene was designed, optimized for expression in Pichia pastoris. This gene was also modified to avoid glycosylation, and cloned into the vector pPICZαA under the control of the methanol inducible ...
Subjects
Medical Subject Headings: parasitic diseases
free text keywords: Carbonic anhydrase, Plasmodium, Pichia pastoris, expression system, Molecular biology, Molekylärbiologi
Related Organizations

1. Assemble the column according to the manufacturer's instructions.

2. Completely resuspend a 50% Ni-NTA Superflow slurry and transfer 2 µl slurry to the column.

3. Allow the resin to settle. Do not allow resin to dry. If this should occur, resuspend resin in lysis buffer and repack the column.

4. Equilibrate column with 6 column volumes of lysis buffer.

5. Transfer the slurry to 50 ml sample and mix gently by shaking (100 rpm) at room temperature for 60 min.

6. Load the sample-Ni-NTA mixture into the column and collect the column flow-through.

7. Wash with 5 column volumes lysis buffer; collect wash fractions for SDS-PAGE analysis.

8. Wash with 5 column volumes wash buffer; collect wash fractions for SDS-PAGE analysis.

9. Elute the protein with 12 column volumes elution buffer. Collect the eluate in fractions of 1 ml size and analyze by SDS-PAGE.

50 mM NaH2PO4 (6.90 g NaH2PO4·H2O (MW 137.99 g/mol)) 300 mM NaCl (17.54 g NaCl (MW 58.44 g/mol)) 10 mM imidazole (0.68 g imidazole (MW 68.08 g/mol)) 50 mM NaH2PO4 (6.90 g NaH2PO4·H2O (MW 137.99 g/mol)) 300 mM NaCl (17.54 g NaCl (MW 58.44 g/mol)) 20 mM imidazole (1.36 g imidazole (MW 68.08 g/mol)) 50 mM NaH2PO4 (6.90 g NaH2PO4·H2O (MW 137.99 g/mol)) 300 mM NaCl (17.54 g NaCl (MW 58.44 g/mol)) 250 mM imidazole (17.00 g imidazole (MW 68.08 g/mol))

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