Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain

Abstract

Background: The expression of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted E. coli strain.

Results: The expression of eight plant proteins in Escherichia coli BL21(DE3)-pLysS and BL21(DE3)-CodonPlus-pRIL was systematically studied. The CodonPlus strain contains extra copies of the argU, ileY, and leuW tRNA genes, which encode tRNAs that recognize the codons AGA/AGG, AUA and CUA, respectively (RIL codons). The level of expression and solubility of the recombinant proteins were analyzed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. We found that for all proteins the solubility was at least 25% in the BL21(DE3)-pLysS strain. However, when expressed in the BL21(DE3)-CodonPlus-pRIL strain, proteins having more than 5% of amino acids coded by RIL codons were localized mainly in the insoluble fraction. Also, their expression caused retarded growth and low cell yield in the codon bias-adjusted strain at all temperatures tested. On the contrary, the solubility of proteins containing less than 5% of amino acids coded by RIL codons remained unchanged in both strains and their expression caused no effect on cell growth.

Conclusion: Our results show that the expression of heterologous proteins coded by high RIL codon content coding sequences in a codon bias-adjusted strain is detrimental for their solubility. Our data support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to protein misfolding and aggregation. This stresses the importance of strain selection according to codon content in any scheme where a large amount of biologically active product is desirable.

Figure 1

Effect of protein expression on CP cells growth. Cells carrying each plasmid were grown at 25°C for 6 h as described in Methods. Blue bars show the percentual change in final OD₆₀₀ while red bars show percentual change in fresh cell weight of the induced culture vs the uninduced culture. Each bar represents the mean (plus error bars) of three independent experiments.

Figure 2

Protein expression in whole lysates. SDS-PAGE and Western blots of total cell proteins. BL uninduced and induced cultures: lane 1 and 2. CP uninduced and induced cultures: lane 3 and 4. Each coding sequence product is marked with a red arrow. Numbers below each blot indicate the fold increase in protein expression in the CP strain compared with the BL strain. The polyacrylamide percentage of each gel was as follow: 10% ClpC2 and ClpD, 12% FNR, ClpP4 and ClpR2 and 15% Fd, ClpT1, dsRBD2 and Trx. To fulfil linearity and detection limits for the inmunodetection method, a fraction of sample loaded in Coomassie Blue stained gels were loaded in Western blots as follows: Fd and FNR (lane 4), 20%; ClpC2, ClpD and dsRBD2 (lane 2), 20%; ClpC2, ClpD, ClpR2 and dsRBD2 (lane 4), 10%.

Figure 3

Distribution of each protein in the soluble and insoluble fractions. SDS-PAGE and Western blots of soluble and insoluble fractions from induced BL (lanes 1 and 2) and CP (lane 3 and 4) cultures. Lane 1 and 3 are supernatants obtained by centrifugation of whole lysates at 10,000 g for 30 min. Lane 2 and 4 are the obtained pellets resuspended with the same volume of buffer as the supernatants. Bar plots show the percentual amount of target protein in each fraction for both strains (blue: supernatant, red: pellet). To fulfil linearity and detection limits for the inmunodetection method, a fraction of sample loaded in Coomassie Blue stained gels were loaded in Western blots as follows: FNR (lane 4), 20%; ClpC2, ClpD and ClpR2 (lane 4), 10%.

Figure 4

Fold change in soluble protein volumetric yield. Fold change represents the log₂ of the ratio of the volumetric yield of each protein (in mg per liter of soluble protein) expressed in the CP strain and the volumetric yield of the same protein expressed in the BL strain. Protein expressed at a constant level (ratio of 1) has a log2(ratio) equal to zero, which can be seen as "no change".

Figure 5

Change in solubility of target proteins as a function of RIL codon content. Change in solubility indicates the difference of the percentage of soluble protein in the CP strain with respect to the percentage of soluble protein in the BL strain. Blue dots, L-RIL proteins; red dots, H-RIL proteins; green dot, thioredoxin.