The cell-free protein biosynthesis--applications and analysis of the system.
The in vitro protein biosynthesis has the potentials to become a powerful technology for biochemical research. Beside the determination of structure and function the in vitro evolution of proteins is also of great interest. The system described was used to produce bovine heart fatty acid binding protein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and without fusion of the Strep-tag II affinity peptide. The proteins were purified after and during protein biosynthesis by using a StrepTactin Sepharose matrix. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the protein was observed. The in vitro evolution of proteins is feasible by means of ribosome display. The selection of a specific mRNA coding for a shortened FABP with a N-terminal His-tag via the accompanying protein property was shown. Goal of the selection was to bind the FABP via the His-tag on Ni(II)-IDA-agarose. After nine cycles of transcription, translation, affinity selection and RT-PCR the protein with the His-tag could be enriched 10(8)-fold. In order to correlate a possible relationship between changes in protein population and biological function studies were initiated in which 2-dimensional protein patterns of the total in vitro system were compared after 0 and 2 h reaction time. The very interesting findings are that a number of proteins disappear, while others are newly formed during protein synthesis.
Lamla
T
T
Institut für Biochemie, Freie Universität Berlin, Germany.
Mammeri
K
K
Erdmann
V A
VA
D000818:Animals; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002417:Cattle; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D050556:Fatty Acid-Binding Proteins; D066298:In Vitro Techniques; D009206:Myocardium / Q000378:metabolism; D009363:Neoplasm Proteins; D019151:Peptide Library; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D019151:Peptide Library; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase