Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system.
In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.
Kim
Tae-Wan
TW
School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-742, Republic of Korea.
Keum
Jung-Won
JW
Oh
In-Seok
IS
Choi
Cha-Yong
CY
Park
Chang-Gil
CG
Kim
Dong-Myung
DM
D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D003362:Cost-Benefit Analysis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000254:growth & development; D005240:Feasibility Studies; D014176:Protein Biosynthesis; D046188:Protein Modification, Translational; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D012269:Ribosomal Proteins; D015500:Chloramphenicol O-Acetyltransferase