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 (Tm p) has become more widespread and several Tmp-resistant (Tmp(r)) dihyd rofolate reductases (DHFRs) have been described from Gram-negative bac teria. In staphylococci, however, only one Tmp(r) DHFR (type S1 DHFR) has been found so far, and this is located on transposon Tn4003. To he lp understand the mechanism of resistance, we are interested in determ ining the 3-D structure of the recombinant enzyme produced in Escheric hia coli. However, the production level of the type S1 DHFR was very l ow 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-le ngth enzyme was produced. We were able to increase the expression leve l 20-fold by changing 18 N-terminal codons and to eliminate the intern al start of translation. In addition, through molecular modelling and subsequent site-directed mutagenesis to replace two amino acids, we co nstructed a biochemically similar but soluble derivative of the type S 1 DHFR that, after production in E.coli, resulted in a 264-fold increa se in DHFR activity. The highly overproduced enzyme was purified to ho mogeneity, characterized biochemically and crystallized.