Rare codon priority and its position specificity at the 5′ of the gene modulates heterologous protein expression in Escherichia coli

• Published in: Biochemical and biophysical research communications 2008-11, Vol.376 (4), p.647-652
• Main Authors: Thangadurai, Chinnathambi, Suthakaran, Pichaimuthu, Barfal, Pankaj, Anandaraj, Balaiah, Pradhan, Satya Narayan, Ramalingam, Subramanian, Murugan, Vadivel
• Format: Article
• Language: English
• Subjects: 5' Flanking Region; AGG codon; Base Sequence; Codon - genetics; DNA Mutational Analysis; E.coli; Escherichia coli; Escherichia coli - genetics; Gene Expression; Genetic Engineering; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; Over-expression; Protein Biosynthesis - genetics; Rare codon; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Streptococcus - enzymology; Streptococcus - genetics; Streptococcus equisimilis; Streptokinase; Streptokinase - genetics
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2008.09.024

Rare codons and their effects in heterologous protein expression in Escherichia coli were addressed by many investigators. Here, we propose that not all rare codons of a foreign gene have negative effect but selective codon among them and its specific position in the downstream of the start codon modulates the expression. In our study, streptokinase (47 kDa), encoded by skc gene of Streptococcus equisimilis was expressed in E.coli. The analysis of relative codon frequency of skc gene in E.coli reveals the presence of 30% of rare codons in it. Nevertheless, E.coli managed to yield over-expression of this target protein. To explore the codon bias in expression, we have introduced the selective AGG codon at different positions of skc gene such as +2,+3,+5,+8,+9 and +11. The results revealed that at +2 position “AGG” aided over-expression while shifting to +3 and +5 positions it rendered nil expression. In contrary, shifting of AGG codon to later positions like +9 and +11 the inhibitory effect was reversed and resulted in over-expression. The effect of ‘AGG’ rare codon was further studied in GFP expression. In conclusion, besides the choice of rare codons, their precise positions in the foreign gene dictate the level of protein expression.