Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells

Abstract

Insect cells are useful for the high-yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell-based system for incorporating non-natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNA(Tyr) in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl-tRNA synthetase specific for 3-iodo-L-tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non-natural amino acids, and the "transient" method generated a sevenfold higher yield. By this method, 4-azido-L-phenylalanine was incorporated into human interleukin-8 at a specific site. The yield of the azido-containing IL-8 was 1 microg/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger-Bertozzi reaction.

Figure 1

Chemical structures of l-tyrosine and its derivatives.

Figure 2

Development of the expression system for prokaryotic suppressor tRNA^Tyr in S2 cells. A: The three expression systems for the bacterial suppressor tRNA^Tyr molecules. “Dm” means D. melanogaster. “TTTTT” indicates the transcription terminator. B: The relative amber suppression efficiencies of the three expression systems, measured in terms of the β-gal activity in the transiently transfected S2 cells. The bar above “vector” shows the β-gal activity with neither LacZ(Am91), iodoTyrRS-ed, nor suppressor tRNA^Tyr. The bar above “–tRNA” shows the β-gal activity without suppressor tRNA^Tyr. The white bar shows 5% of the β-gal activity due to the wild-type LacZ transiently expressed in the cells. C: A map of the plasmid pAcEYR/iodoTyrRS-ed. D: The amber suppression due to the U6 promoter system for expressing the E. coli suppressor tRNA^Tyr. “–tRNA,” “–aaRS,” and “–IY” mean the absence of the suppressor tRNA^Tyr, iodoTyrRS-ed, and 3-iodo-l-tyrosine, respectively. Each bar represents the average of three independent experiments throughout this figure.

Figure 3

Enrichment of the stably transfected S2 cells. (A) Schematic illustration of the applied procedure. For details, refer to Materials and Methods section. (B–E) S2 cells harboring pIY-LacZ(Am91) (B, D) and pIY-LacZ (C, E) were stained with X-gal at the selection steps indicated in panel A. The percentage of stained cells is shown above each panel.

Figure 4

Production of IL-8 variants containing 3-iodo-l-tyrosine and 4-azido-l-phenylalanine in S2 cells. (A, B) SDS-polyacrylamide electrophoresis of the purified IL-8 and its variants with the non-natural amino acids. Five- and 10-percent amounts of the obtained wild-type IL-8 were applied for comparison. The product of the amber mutant IL-8 gene was obtained in the presence of 3-iodo-l-tyrosine (+IY) and 4-azido-l-phenylalanine (+AzF), while it was scarcely obtained in the absence of these amino acids (–IY, –AzF). (C) A fluorescent image of the SDS-polyacrylamide gel of panel B, obtained with an LAS-1000 image analyzer (Fujifilm, Japan).

Figure 5

MALDI-TOF analysis of the IL-8 derivatives before (A) and after (B) the labeling reaction with a triarylphosphine-biotin conjugate. The peaks X, Y, and Z correspond to the IL-8 molecules with 4-aminophenylalanine and AzF, and the biotin-labeled IL-8 molecule, respectively.