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Codon Optimization Protocol


Codon Optimization Protocol

Codon optimization is one of the key steps in achieving the high level expression of target gene. There are some key factors for consideration including transcription efficiency, translation efficiency, gene synthesis and protein folding.

Some necessary consideration factors in codon optimization

1. tRNA abundance

Owing to different tRNAs identify several synonymous codons and the content of these synonymous tRNAs are different, therefore, the efficiency of translation also depends on the number of tRNA. Preferred codons are those that can base pair optimally with the most abundant tRNA. Generally this involves Watson - Crick pairing or, when bases are modified in the tRNA, some modifications in optimal binding occur.

2. Codon usage bias

There are obvious biases of synonymous codon in bacteria, Escherichia coli, yeast and some expression systems of higher biological. This can directly affect the efficiency of translation. So when one gene is expressed in a heterologous system, the codon usage bias should be taken into account.

3. Complete mRNA structure

Next to 5’UTR and 3’UTR, the complete mRNA structure also has significant impact on the process of protein translation. Recently, from the systematic study of major factors which affect the protein expression, we know that the folding of mRNA, especially the mRNA structure near the RBS, plays an important role in protein expression. For example, the level of protein expression can be greatly improved by reducing the secondary structure near the start codon.

4. The theory of gene design

Codon adaptation index (CAI)

CAI measures the proximity between the codon which is used by one gene and used in gene with high expression. CAI is between 0 and 1. The closer that the CAI is to 1, the higher level of gene expression is. In general, CAI>0.8 means that gene expression has a high level.

Codon correlation index (CCI)

Researches show that codon correlation of specific host exert considerable influence on the expression of heterologous genes. Codon correlation, such as codon usage, double codon usage, dynamic of translation phase and GC content, describe an integrated evolutionary environment characteristic of species codon selection.

Codon optimization can solve the problem of protein expression as follow:

  • Unable to be expressed in heterologous systems;
  • Expression level was very low;
  • Unable to fold properly;
  • Lose functional activity.

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