BALTIMORE—The widespread use of protein chips has largely been limited by the need for large-scale protein purification to generate a broad spectrum of proteins in a given cell type. Researchers at Johns Hopkins School of Medicine, however, have recently described their efforts to produce protein chips using on-chip gene expression and polypeptide immobilization.
As they describe in Nature Biotechnology, the researchers relied on the fact that when a ribosome reaches duplex nucleic acid, it pauses just long enough for a puromycin molecule to enter the polypeptide synthesis site and become covalently bound to the nascent protein chain. Thus, in one scheme, the researchers attached mRNAs to silicon chips via streptavidin-biotin links. They then annealed a second short RNA primer to slow peptide synthesis and attached a puromycin-labeled oligo to the chip to capture the new peptide.
The researchers found that whether they were producing peptide tag sequences or full-length proteins, they could capture about 0.8 fmol protein per spot (or about 40,000 molecules). Furthermore, the captured peptides could be detected with anti-peptide antibodies and were capable of performing their normal biological functions.