title: Rare codon priority and its position specificity at the 5' of the gene modulates heterologous protein expression in Escherichia coli.
abstract: 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.
authors.affiliation: Genetic Engineering Unit, Centre for Biotechnology, Sadar Patel Road, Anna University, Guindy, Chennai-600 025, India.
authors.forename: Chinnathambi
authors.initials: C
authors.lastname: Thangadurai
authors.forename: Pichaimuthu
authors.initials: P
authors.lastname: Suthakaran
authors.forename: Pankaj
authors.initials: P
authors.lastname: Barfal
authors.forename: Balaiah
authors.initials: B
authors.lastname: Anandaraj
authors.forename: Satya Narayan
authors.initials: SN
authors.lastname: Pradhan
authors.forename: Subramanian
authors.initials: S
authors.lastname: Ramalingam
authors.forename: Vadivel
authors.initials: V
authors.lastname: Murugan
mesh_terms: D024506:5' Flanking Region; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics; D013300:Streptokinase / Q000235:genetics
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D013300:Streptokinase