Mian Zhou, Jinhu Guo, Joonseok Cha, Michael Chae, She Chen, Jose M. Barral, Matthew S. Sachs and Yi Liu
School of Biotechnology, East China University of Science and Technology, Shanghai, China
During heterologous protein expression, the aim is to increase expression levels without altering native activities. Gene expression is mostly known to be regulated at the transcriptional level, while protein function can be regulated post-translationally. However, as a co-translational mechanism, the role of codon usage in regulating protein expression and function is less well studied.
In many organisms, there is a bias for preferred codons and it is thought to be a mechanism to enhance expression for highly expressed genes. For example, Neurospora crassa prefers to use G/C rather than A/T at the 3rd position of codons in highly expressed genes. FREQUENCY (FRQ) is a key component in the negative feedback loop of Neurospora crassa circadian clock. Compared with other genes, frequency (frq) exhibits a very non-optimal codon usage. It's not clear whether this "poor" codon usage has any biological significance.
To test the role of codon usage in frq, here we made a series of codon optimized frq constructs, and introduced them into a frq null strain. We found that strains carrying the N-terminal optimized frq exhibited severe circadian clock phenotypes, with higher FRQ protein levels but similar frq mRNA levels compared with wild-type. Surprisingly, shown by both trypsin sensitivity assay and freeze-thaw assay, FRQ proteins in these strains were less stable. Besides, they were defective in binding their WCC partners, and optimized FRQ had a compromised function to support WC-1 and WC-2 levels in the positive feedback loop, suggesting that codon optimization also influences protein function even though its sequence is unchanged. Taken together, codon usage of frq regulates its protein expression, folding and function. Therefore a scientific analysis is needed during codon optimization design when expressing heterologous proteins.
Keywords: Codon usage, expression, folding.