Posttranslational modifications in an insect cell-free protein synthesis system and their identification by MALDI-TOF MS

MS / MS analysis N-myristoylation Summary We have established a cell-free protein synthesis system (Transdirect insect cell) derived from Spodoptera frugiperda 21 insect cells [1]. This cell-free system has high protein productivity, and therefore it is expected to be sufficient to perform gene expression analyses including not only the measurement of enzymatic activity and western blotting, but also investigation of posttranslational modifications. In this study, several posttranslational modifications in the insect cell-free protein synthesis system were confirmed and identified by MALDI-TOF MS [2, 3, 4].

In general, protein N-myristoylation results from the co-translational addition of myristic acid to a Gly residue after removal of the initiating Met. The requirement for Gly is absolute, and Ser or Thr at position 6 is preferred.

Summary
We have established a cell-free protein synthesis system (Transdirect insect cell) derived from Spodoptera frugiperda 21 insect cells [1]. This cell-free system has high protein productivity, and therefore it is expected to be sufficient to perform gene expression analyses including not only the measurement of enzymatic activity and western blotting, but also investigation of posttranslational modifications. In this study, several posttranslational modifications in the insect cell-free protein synthesis system were confirmed and identified by MALDI-TOF MS [2, 3, 4].

S-S bond formation
Human lysozyme (h-LYZ), which contains four disulfide bonds was expressed in the insect cell-free system under following conditions: a) reducing condition by DTT, b) non-reducing condition, c) non-reducing condition in addition of GSH, GSSG, and PDI. Analysis of the synthesized proteins were carried out using FluoroTectTM (Promega Co.). The synthesized proteins were separated by centrifugation, and soluble and precipitated fractions were analyzed by SDS-PAGE and fluoroimager (Fig. 1A). The synthesized proteins were almost in the soluble fraction under such conditions. The protein synthesis efficiencies were similar to each other. However, lysozyme activity was detected strongly in the h-LYZ synthesized under non-reducing condition in addition of GSH, GSSG, and PDI (Fig. 1B).
Using the optimized condition where lysozyme activity was expressed at the highest levels, h-LYZ was synthesized, purified by affinity column chromatography, and the purity was judged on SDS-PAGE by staining with CBB (Fig. 2). The h-LYZ ran as almost single band having molecular mass of about 17 kDa. The final yield of h-LYZ was approximately 30 μg from 1 ml reaction mixture. The specific activity of the h-LYZ synthesized in vitro was similar to that of the wild type h-LYZ (SIGMA) prepared from human milk (data not shown). Furthermore analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed (data not shown). The insect cell-free protein synthesis system can be an effective tool for synthesizing active proteins containing disulfide bonds.

N-terminal protein modifications
Epitope-tagged truncated human gelsolin (tGelsolin), which is natural N-myristoylated protein, was synthesized using the insect cell-free protein synthesis system with or without addition of a specific substrate for N-myristoylation, such as myristoyl-CoA (Fig. 4). When myristoyl-CoA was added to the translation reaction mixture, a peak corresponding to the Nmyristoylated tryptic peptide was clearly observed, whereas this peak was hardly detected when myristoyl-CoA was not added (Fig. 6). The peptide peak was identified as the Nmyristoylated peptide fragment by tandem MS analysis using MALDI-QIT-TOF MS (Fig. 7A). We also constructed the Gly-2 to Ala (G2A) mutant, in which the N-myristoylation motif was disrupted, and analyzed by the same strategy. Nmyristoylation did not occur on the G2A mutant, whereas this mutant was found to be N-acetylated after removal of the initiating Met (Fig. 6&7B).
A combination of the cell-free protein synthesis system with MS is an effective strategy to analyze N-terminal protein modifications.