Amplification of protein expression in a cell free system

Abstract

Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNAfol, the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polymerase chain reaction (PCR) and (ii) in vitro RNA transcription by T7 RNA polymerase, followed by (iii) translation of the run-off transcripts in a rabbit reticulocyte system. The protein product had the expected size (18 kDa) and catalyzed the NADPH-dependent reduction of 7,8-dihydrofolic acid to 5,6,7,8-tetrahydrofolic acid as efficiently as authentic DHFR. Potential applications of the strategy include large scale production of enzymes containing synthetic amino acids and facilitation of the characterization of the function of genes encountered in genomic mapping studies.

Probing the Role of an Active Site Aspartic Acid in Dihydrofolate Reductase

“…This system was employed previously for the overexpression of DHFR according to a strategy that coupled the polymerase chain reaction with transcription and translation; the derived protein was fully competent as a catalyst relative to wild-type DHFR isolated from E. coli. 5 Because the elaboration of proteins in cell free systems requires the addition of protein factors in greater total abundance than the protein species actually synthesized, it is essential to have some strategy for purification of the derived proteins. In the present case, a hexahistidine moiety included as a fusion peptide at the N-terminus was able to bind to a Ni-NTA agarose affinity resin; subsequent elution with a high salt buffer containing 10 mM imidazole effected elution of the purified protein.…”

“…4 We have previously described the synthesis of E. coli DHFR in a cell free system that was amenable to overexpression, and which produced wild-type DHFR shown to be fully competent catalytically. 5 Herein, we demonstrate that by introducing a nonsense (UAG) codon at a specific position in the DHFR mRNA, it is possible to elaborate modified DHFRs by inclusion of misacylated suppressor tRNA CUA s in the protein biosynthesizing system (Scheme 1). Presently, we describe the synthesis of seven novel DHFRs containing aspartic acid analogues at position 27.…”

RNA stem-loop enhanced expression of previously non-expressible genes

“…Recombinant plasmids were ampli®ed by one-step PCR to generate templates for`run-off' transcription (27) and for in vivo protein synthesis.…”

Charging of tRNA with non‐natural amino acids at high pressure

“…The preparation of tRNA charged with non-natural amino acid is a critical step in the synthesis of modified protein. All methods of preparing aa-tRNA charged with non-native amino acids are complicated and timeconsuming [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. In this study, we developed a general method of tRNA aminoacylation using any amino acid.…”

“…The utility of mischarged tRNAs has been expanded by developing chemical acylation of the unprotected dinucleotide pCpA, followed by enzymatic ligation to the 3′‐terminus of truncated tRNA using T4 RNA ligase. This approach has a low acylation yield of 3–4%[13,14]. An improved version of the method, based on the acylation of fully protected 5′pCpCpA resulted in 26% charging [15].…”