Signal sequence non-optimal codons are required for the correct folding of mature maltose binding protein.
Non-optimal codons are generally characterised by a low concentration of isoaccepting tRNA and a slower translation rate compared to optimal codons. In a previous study, we reported a 20-fold reduction in maltose binding protein (MBP) level when the non-optimal codons in the signal sequence were optimised. In this study, we report that the 20-fold reduction is rescued when MBP is expressed at 28 degrees C instead of 37 degrees C, suggesting that the signal sequence optimised MBP protein (MBP-opt) may be misfolded, and is being degraded at 37 degrees C. Consistent with this idea, transient induction of the heat shock proteases prior to MBP expression at 28 degrees C restores the 20-fold difference, demonstrating that the difference in production levels is due to post-translational degradation of MBP-opt by the heat-shock proteases. Analysis of the structure of purified MBP-wt and MBP-opt grown at 28 degrees C showed that although they have similar secondary structure content, MBP-opt is more resistant to thermal unfolding than is MBP-wt. The two proteins also exhibit different tryptic fragment profiles, further confirming that they are folded into conformationally different states. This is the first study to demonstrate that signal sequence non-optimal codons can influence the folding of the mature exported protein.
School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Australia.
Yaramah M
YM
Zalucki
Christopher E
CE
Jones
Preston S K
PS
Ng
Benjamin L
BL
Schulz
Michael P
MP
Jennings
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D006358:Hot Temperature; D033902:Periplasmic Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000502:physiology*
D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals