Manipulate Protein Expression In Escherichia Coli By Codon Bias

With clear genetic background, short expression cycle and the ability of large-scale fermentation, Escherichia coli has been scientists’favorite stain for cloning and expression of heterologous proteins. However, as the foreign genes expression system, the translation speed of Escherichia coli is too fast, protein expression level is too high and easy to form inclusions, which often brings very serious negative effects. In addition, in the metabolic engineering of E. coli, to realize the joint coordinated expression of genes, the protein expression level of all the enzymes in metabolic pathway must limit in a certain range. Although there are many mature control systems of gene expression, few of them can reach the demand of metabolic engineering.In this study, rare codons were used to reduced the translation speed and reduced the protein expression level accordingly, and finally a set of modules were got. These modules could down-regulate protein expression level according to the different gradient. And it can be applied in metabolic engineering.The results are as follows:1In the pET expression system, using inorganic pyrophosphatase of Saccharomyces cerevisiae as model, the protein expression level can be down-regulated by adding two and four rare codons. Then we transformed the former plasmid to Rosetta. But when the same plasmids was transformed into Rosetta, the inhibition effect is averted.(Rosetta contains a plasmid carrying corresponding tRNA gene of rare codons.) This preliminary verified that the rare cotton can down-regulated the protein expression.2 DpnI site directed mutation were made on the rare codons based on the result of OR, RR, RRRR. Then screened and validated a series of rare codons, and analysed their relative activity. The protein expression level (from high to low) were OR (100.00),RGGR (75.50),RS (70.61),RGRG (56.35), RR (42.99), RRSR (30.37), RRGR (22.36), RRRG (4.39), RRRS (2.91), RRRR(0). When the OR, RR, RRRR were moved from+21 (number of codons from ATG) to+2, the difference still existed.3 Part of the rare codons such as RR、RRRR、RRRS、RRRG were applied on pBAD expression system. The result was similar to the above result in the pET expression system, though some changes existed. The relative activity of RR changed from 42.99 to 68.57; the relative activity of RRRS changed from 2.91 to 51.74; the relative activity of RR changed from 4.39 to 57.28.4 Added the rare codons (also called codon regulatory elements) before the BglA which the protein expression form is always inclusion body, the total protein expression level all have different degrees of down-regulation. Soluble protein expression from high to low were SS(141.15),G2G2 (139.40),G1G1 (132.17), OR (100.00), RR(65.09), RRSR(37.66), RRGR(20.59). Among them, the solubility of the soluble proteins which added SS and G2G2 was increased 1.4 times, but the total protein did not increase.5 In the process of modifying MEP pathway, the protein expression level of IspG which added rare codons all had different degrees of down-regulation. Correspondingly the output of neurosporene were different. Among them, the protein expression level of RRRR-IspG was the highest one.