FAQspeak

Three common questions about the TNT^® Quick Coupled Rabbit Reticulocyte Lysate Systems^(a)* (Cat.# L1170, L1171, L2080, L208)

How much DNA should I use in the TNT^® Quick Coupled Rabbit Reticulocyte Lysate reaction? What are the DNA template considerations? How much protein can I make?

The standard protocol in the TNT^® Quick Coupled Rabbit Reticulocyte Lysate reaction (#TM045) suggests using 1µg of plasmid DNA. However one can still obtain good results within the range of 0.2-2.0µg of plasmid DNA. Increasing the amount of DNA above 1µg does not appreciably increase the amount of protein produced. For PCR-generated templates, use less starting material (100-800ng for a 500bp product in a 50µl TNT^® Quick reaction for example), as PCR templates are generally much smaller than plasmid templates.

We also offer TNT^® T7 Quick for PCR^(a) (Cat.# L5540).

The amount of protein produced is dependent on the DNA template. There are several elements that can increase the efficiency of translation. For PCR-generated DNA templates, it is important to include at least 20nt downstream of the T7 RNA polymerase promoter to ensure efficient binding of the polymerase to the promoter. It is also important to add a stop codon (usually UAA) to truncated gene products in order to prevent ribosomes from stalling at the ends of linear DNA. In addition, the presence of a hairpin secondary structure in the 5’ untranslated region (UTR) of the template can affect translation efficiency (1). Multiple AUGs in the sequence can also lead to the production of multiple truncated proteins. If using linearized plasmid DNA in the T7 system, avoid the use of restriction enzymes that will yield 3’-overhangs (e.g., Apa I, Apa II, BstX I, Kpn I, Nsi I, Pst I, Sac I, Sac II), as aberrant translation products can be produced (2). Enhanced translation of proteins has been observed when using DNA constructs containing a poly(A) sequence downstream of the gene of interest. Poly(A) sequences are important for mRNA stability and can play a role in translation initiation in Rabbit Reticulocyte Lysate (3).

As a rough guideline, the positive control supplied with the system can produce 200-300ng of protein per reaction.

References

1. Frances, V., Morle, F. and Godet, J. (1992) Identification of two critical base pairings in 5’ untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs. Biochim. Biophys. Acta 1130, 29-37.
2. Schenborn, E. and Mierendorf, R.C. (1985) A novel transcription property of SP6 and T7 RNA polymerases: Dependence on template structure. Nucl. Acids Res. 13, 6223-6236.
3. Jackson, R.J. and Standart, N. (1990) Do the poly(A) tail and 3’ untranslated region control mRNA translation? Cell 62, 15-24.

What are the minimum and maximum size proteins that can be produced using the TNT^® Coupled Rabbit Reticulocyte Lysate Systems?

The smallest protein that can be produced using the system is about 15kDa. Below that, the ubiquitin degradation system present in the Rabbit Reticulocyte Lysate will actively degrade the protein. The largest protein produced using the TNT^® system, that we are aware of, was ~200kDa (1).

Reference

1. Hemmer, O. et al. (1995) Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-1-encoded polyprotein. Virology 206, 362-371.

What is the function of the hemin added to the Rabbit Reticulocyte Lysate?

Hemin is added to the lysate to suppress an inhibitor of the initiation factor elF2a. Without hemin, protein synthesis in the Reticulocyte Lysate System would cease after a short incubation period (1).

References

1. Jackson, R. and Hunt, T. (1983) Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Meth. Enzymol. 96, 50-74.

^(a)For Laboratory Use.

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