Fusion enhanced protein accumulation

Patel, J., Zhu, H., Menassa, R., Gyenis, L., Rich-man, A., and Brandle, J. (2007). Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves. Transgenic Res. 16,239-249. 

To conclude this mutational pathway, PMIL was sculpted by 12 rounds of directed evolution, in which it accumulated 22 mutations (8 silent) throughout the entire fusion gene. Beneficial mutations that enhanced secretion or activity were located in the signal prepro-leader (5 mutations) and the mature protein (7 mutations), respectively. Significantly, only two mutations located in the second coordination sphere of the T1 copper site conferred tolerance to blood. Therefore, the re-specialization required to adapt the PMIL to such inclement conditions affected only 0.4% of the amino acid sequence. 

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