Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase.
In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread and several Tmp-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from Gram-negative bacteria. In staphylococci, however, only one Tmpr DHFR (type S1 DHFR) has been found so far, and this is located on transposon Tn4003. To help understand the mechanism of resistance, we are interested in determining the 3-D structure of the recombinant enzyme produced in Escherichia coli. However, the production level of the type S1 DHFR was very low and > 95% of the total recombinant protein accumulated in inclusion bodies. Furthermore, as a result of an internal start of translation, a truncated derivative of the enzyme that copurified with the full-length enzyme was produced. We were able to increase the expression level 20-fold by changing 18 N-terminal codons and to eliminate the internal start of translation. In addition, through molecular modelling and subsequent site-directed mutagenesis to replace two amino acids, we constructed a biochemically similar but soluble derivative of the type S1 DHFR that, after production in E.coli, resulted in a 264-fold increase in DHFR activity. The highly overproduced enzyme was purified to homogeneity, characterized biochemically and crystallized.
F. Hoffmann-La Roche Ltd., Basel, Switzerland.
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Dale
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Broger
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Langen
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D'Arcy
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Stüber
D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D003460:Crystallization; D004926:Escherichia coli / Q000378:metabolism; D005493:Folic Acid Antagonists; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012995:Solubility; D013211:Staphylococcus aureus / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D014295:Trimethoprim / Q000494:pharmacology; D014296:Trimethoprim Resistance
D001426:Bacterial Proteins; D005493:Folic Acid Antagonists; D011993:Recombinant Fusion Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase