High-throughput construction and screening of protein libraries by cell-free systems

A novel, biological-cloning-independent strategy for construction of protein libraries has been developed and demonstrated experimentally. A pool of genes is extensively diluted to give one molecule of DNA per well. Instead of transformation of living cells, each individual molecule is amplified separately by polymerase chain reaction (single-molecule PCR) yielding a DNA library named 'PCR library'. Subsequently, the PCR library is directly transformed into a protein library by means of in vitro coupled transcription/translation. Amounts of DNA produced by the single-molecule PCR were equal and uniformity of amounts of successively in vitro synthesized proteins, which are critical for quantitative comparison among clones in protein library, were better than those of the classical in vivo expression system. Libraries of single-chain Fv, and bacterial lipase, the green florescent protein of Aequorea victoria have been constructed by the system to demonstrate its applicability. Application of the strategy described for high-throughput, generation and screening of protein libraries will be discussed.