CHAPTER 1 Cell-Free Protein Synthesis 3 ALEXANDER S. SPIRIN CHAPTER 1 Cell-Free Protein Synthesis 1 ALEXANDER S. SPIRIN* Abbreviations NTP nucleoside triphosphates aminoacyl-tRNA synthetases ARSes PEP phosphoenol pyruvate CP creatine phosphate AcP acetyl phosphate PK pyruvate kinase CK creatine kinase AcK acetyl kinase ME mercaptoethanol DTT dithiotreitol DHFR dihydrofolate reductase CAT chloramphenicol acetyltransferase GFP green fluorescent protein IL-2 and IL-6 interleukin-2 and interleukin-6 TMV tobacco mosaic virus Introduction: Prehistory of Cell-Free Translation Systems As early as the beginning of the 1950s, several groups independently demonstrat- ed that protein synthesis does not require the integrity of the cell and can contin- ue after cell disruption. Thus, disrupted cells or their isolated fractions were reported to be capable of synthesizing proteins (Borsook 1950; Winnick 1950a, 1950b; Siekevitz and Zamecnik 1951; Siekevitz 1952; Peterson and Greenberg 1952; Khesin 1953; Gale and Folkes 1954). In the meantime, ribonucleoprotein particles were observed and identified in cells (Palade 1955) and then isolated from cells and studied with respect to their physicochemical properties (Chao and Schachman 1956; Ts'o et al. 1956; Peterman and Hamilton 1957; Tissieres and Watson 1958; Tissieres et al. 1959; see also papers in Roberts 1958). The protein- * Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia Tel. !Fax: 007(095)924-0493, e-mail: spirin@vega. protres. ru Introduction synthesizing ability of these particles was experimentally proved (Littlefield et al. 1955; Littlefield and Keller 1957; McQuillen et al. 1959). The word "ribosome" was proposed to designate the protein-synthesizing ribonucleoprotein particles.