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 Ig alpha, 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 Ig alpha 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. Published by Elsevier Inc.