In Vitro Translation: The Basics The in vitro synthesis of proteins in cell-free extracts is an important tool for molecular biologists. It has a variety of applications, including the Optimization of protien expression, localization of mutations through synthesis of truncated gene products, protein folding studies etc.

Cell-Free Expression Systems The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes and Escherichia coli. All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg2+, K+, etc.). There are two approaches to in vitro protein synthesis based on the starting genetic material: RNA or DNA. Standard translation systems, such as reticulocyte lysates and wheat germ extracts, use RNA as a template; whereas "coupled" and "linked" systems start with DNA templates, which are transcribed into RNA then translated.

Rabbit Reticulocyte Lysate Rabbit reticulocyte lysate is a highly efficient in vitro eukaryotic protein synthesis system used for translation of exogenous RNAs (either natural or generated in vitro). These immature red cells, contain adequate mRNA, as well as complete translation machinery, for extensive globin synthesis. The endogenous globin mRNA can be eliminated by incubation with Ca2+-dependent micrococcal nuclease, which is later inactivated by chelation of the Ca2+ by EGTA.

Important Elements For Translation There are some significant differences between prokaryotic and eukaryotic mRNA transcripts. Typically, eukaryotic mRNAs are characterized by two post-transcriptional modifications: a 5'-7 methyl-GTP cap and a 3' poly(A) tail. Both modifications contribute to the stability of the mRNA by preventing degradation. Additionally, the 5' cap structure enhances the translation of mRNA by helping to bind the eukaryotic ribosome and assuring recognition of the proper AUG initiator codon. This function may vary with the translation system and with the specific mRNA being synthesized. The consensus sequence 5'-GCCACCAUGG-3', also known as the "Kozak" sequence, is considered to be the strongest ribosomal binding signal in eukaryotic mRNA.

In eukaryotes, the Kozak sequence A/GCCACCAUGG, which lies within a short 5' untranslated region, directs translation of mRNA.. In contrast to the E. coli ribosome, which preferentially recognizes the Shine-Dalgarno sequence . The +1 A is the first base of the AUG initiator codon (shaded) responsible for binding of fMet-tRNAfMet. The underline indicates the ribosomal binding site sequence, which is required for efficient translation.

Trichloro acetic acid for protien precipitation Standard in Vitro Translation Procedure Using Rabbit Reticulocyte Lysate

Linked Transcription:Translation The "linked" system is a two-step reaction, based on transcription with a bacteriophage polymerase followed by translation in the rabbit reticulocyte lysate or wheat germ lysate. Because the transcription and translation reactions are separate, each can be optimized to ensure that both are functioning at their full potential. Conversely, many commercially available eukaryotic coupled transcription:translation systems have compromised one or both reactions so that they can occur in a single tube. Thus, yield is sacrificed for convenience.

Linked in Vitro Transcription and Translation Procedure Using Rabbit Reticulocyte Lysate

Very high protein yield Comparison of in vitro (cell-free) protein expression systems. Advantages and disadvantages of existing extract-based systems for human recombinant protein synthesis. Selection of a cell-free expression system should consider the biological nature of the protein, application, and the template used for protein expression. System Advantages Disadvantages E. coli Very high protein yield Many eukaryotic proteins insoluble upon expression Eukaryotic co- and post-translational modifications not possible Codon usage is different from eukaryotes Rabbit Reticulocyte (RRL) Mammalian system Modifications are possible Sensitive to additives Protein glycosylation not possible Co-expression of off-target proteins Wheat germ Translation of large proteins possible Devoid of off-target endogenous mammalian proteins High protein yield Mammalian co- and post-translational modifications are not possible Premature termination of products Insect No endogenous mammalian proteins Certain forms of protein glycosylation possible Non-mammalian Human Human system Co- and post-translational modifications are possible Synthesis of functional proteins Possible to make VPLs (virus-like particles) Lower yields than E. coli New system