Influence of the second amino acid on recombinant protein expression

Abstract

Factors affecting protein expression have been intensely studied to the benefit of recombinant protein production. Through mutational analysis at the +2 amino acid position of recombinant Igα, we examined the effect of all 20 amino acids on protein expression. The results showed that amino acids at the +2 position affected 10-fold in the recombinant protein expression. Specifically, Ala, Cys, Pro, Ser, Thr, and Lys at the +2 position resulted in significantly higher expression of recombinant Igα than other amino acids, while Met, His and Glu resulted in greatly reduced protein expression. This expression difference depended on the amino acid instead of their codon usage. Consistent with the mutational results, a statistically significant enrichment in Ala and Ser at the +2 position was observed among highly expressed Escherichia coli genes. This work suggests a general approach to enhance protein expression by incorporating an Ala or Ser after the initiation codon.

Fig. 1

Initial results indicating that substituting a serine at the second amino acid position results in higher protein expression in E. coli. The lanes represent the un-induced (-) and induced (+) samples of Igα, from left to right, in BL21 (DE3), BL21 Rosetta cells and Leu to Ser mutation in BL21 Rosetta cells. The low molecular weight standard is shown on the left.

Fig. 2

Coomassie blue stained SDS gels to examine the expression of all amino acid substitutions. (A, B) Multiple clones of individual mutations (Ser, Val, Glu, His and Met) were examined by SDS-gel for their clonal variation. The bands for the 40 kDa endogenous bacterial protein and Igα are indicated. Un-induced (-) and IPTG induced (+) lanes are indicated. (C,D) Expression of mutant Igα at the +2 position with their mutations indicated by single letter amino acid code. (E) The normalized expressions of each Igα mutation. Intensities were derived from three separate experiments. (F) Expressions of CXCL10 with Val (wildtype), Ala and Ser as +2 residues.

Fig. 2

Coomassie blue stained SDS gels to examine the expression of all amino acid substitutions. (A, B) Multiple clones of individual mutations (Ser, Val, Glu, His and Met) were examined by SDS-gel for their clonal variation. The bands for the 40 kDa endogenous bacterial protein and Igα are indicated. Un-induced (-) and IPTG induced (+) lanes are indicated. (C,D) Expression of mutant Igα at the +2 position with their mutations indicated by single letter amino acid code. (E) The normalized expressions of each Igα mutation. Intensities were derived from three separate experiments. (F) Expressions of CXCL10 with Val (wildtype), Ala and Ser as +2 residues.

Fig. 3

Amino acid enrichment at the 2^nd -5^th and 40^th positions for 200 highly expressed and 200 randomly chosen proteins from E. coli. The frequency of an amino acid appearing at a given position is defined as the ratio between the number of times that amino acid occurred at that position and the total number of amino acids.

Fig. 3

Amino acid enrichment at the 2^nd -5^th and 40^th positions for 200 highly expressed and 200 randomly chosen proteins from E. coli. The frequency of an amino acid appearing at a given position is defined as the ratio between the number of times that amino acid occurred at that position and the total number of amino acids.

Fig. 3

Amino acid enrichment at the 2^nd -5^th and 40^th positions for 200 highly expressed and 200 randomly chosen proteins from E. coli. The frequency of an amino acid appearing at a given position is defined as the ratio between the number of times that amino acid occurred at that position and the total number of amino acids.

Fig. 4

Isocodon effect on protein expression. (A) Isocodons for alanine mutation are shown in Lane 1-4 for GCG, GCA, GCC, GCU, respectively. Lane 5 is the wild type protein with leucine at the second position. (B) The expression using six serine isocodons are shown in Lane 1-6 for UCG, UCC, UCA, UCU, AGC, AGU respectively. (C) Expression of lysine using both isocodons. Lane 1 is the wild type leucine, Lanes 2 and 3 are AAA and AAG, respectively. (D) Ala, Ser, and Lys codon usage at the +2 position was compared between high expressing genes and randomly selected genes.

Fig. 5

Mutations grouped by their nucleotide usage for the first (A) and second (B) bases of at the +2 amino acid.

Fig. 6

RNA structure prediction of the wild type (A) and alanine (B) mutant. RNA structures were predicted using the RNAfold webserver. The dashed red oval indicates the Shine-Dalgarno sequence, while the AUG initiation codon is located in the red box. (C) The minimum free energy obtained from the RNAfold webserver for each mutation was plotted against normalized expression.