A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility.
Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.
Japrung
D
D
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Science Park, Pathumthani 12120, Thailand.
Chusacultanachai
S
S
Yuvaniyama
J
J
Wilairat
P
P
Yuthavong
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D005816:Genetic Complementation Test / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics*; D002727:Proguanil; D015202:Protein Engineering / Q000379:methods*; D020836:Protein Structure, Quaternary; D011739:Pyrimethamine / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012995:Solubility; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D014227:Triazines / Q000494:pharmacology
D005493:Folic Acid Antagonists; D011993:Recombinant Fusion Proteins; D014227:Triazines; D049452:Green Fluorescent Proteins; C026009:cycloguanil; D013762:Tetrahydrofolate Dehydrogenase; D002727:Proguanil; D011739:Pyrimethamine