Supplemental Figure 5: Subcloning and expression of single domains leads to elevated
protein amounts
Supplemental Figure 5: Subcloning of sequence optimized domains: Gene constructs were expressed using
the sequence-optimized and wildtype coding sequences, respectively, in E. coli BL21(DE3) strains using
autoinduction medium. A, Expression levels for the full length Toll-like receptor 2 (TLR-2, membrane protein)
were visualized after separation of crude lysates by SDS-PAGE and Western blotting using Penta·His antibodies.
Crude lysates of E. coli BL21(DE3) expressing the sequence-optimized C-terminal TIR-domain of TLR-2 and an
empty vector control were analyzed on a SDS gel and subsequently stained with Coomassie Brilliant blue. B,
Expression of the full-length Jak-2 kinase gene and a 297 aa portion of the kinase domain (JH1) using a
sequence-optimized construct and an empty vector control were analyzed on a SDS gel and subsequently stained
with Coomassie Brilliant blue. C, Schematic drawing of the TLR-2 and Jak-2 constructs indicating the size and
boundary of the expressed subdomains.
For those cases where full-length proteins are difficult to purify or show degradation products,
the optimized expression constructs might be useful to express at least single domains of the
protein. Accordingly, subcloning and highly efficient expression of single domains could be
demonstrated for, e. g., the Toll-like receptor 2 (TIR domain) and Jak-2 kinase (JH1 domain).
Thus, our optimization algorithm is not only adjusted to the full length sequence but also turns
out to be successful when only subdomains of a protein are expressed and hence only parts of
the mRNA are transcribed.