proteins by cell-free synthesis: A practical protocol.

Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

In vitro Protein Kinase Assay —BIO-PROTOCOL

The ’tides express combines an extended mass range, compact.
PURExpress ® In Vitro Protein Synthesis PROTEIN EXPRESSION ANALYSIS or in direct activity assays.

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Purification of the target protein can often.
Cayman’s Protein Synthesis Assay Kit is a non-radioactive method for measuring protein synthesis that employs the cell-permeable, alkyne-containing, puromycin.

A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described.
Commercial use of PURExpress® In vitro Protein Synthesis Kit requires a license from New England Biolabs, Inc.


For a detailed protocol and background ..

Results and DiscussionThe preparation of the mRNA template coding for the 10th fibronection type III domain (21 out of 94 amino acids were randomized) began with the transcription from a PCR-DNA library (1012 different sequences) according to the method described in reference (5) (Fig. 1). The freshly transcribed mRNA was annealed to a limiting amount of puromycin-linker carrying a photo-cleavable biotin group for affinity purification on solid-phase (Fig. 2). The hybridized mRNA template-linker complex was then incubated with neutravidin agarose beads followed by washing to remove excess mRNA and transcription buffer components. UV-irradiation of the beads leads to the cross-linking of the two strands by psoralen and to simultaneous cleavage of the biotin-tag. The predetermined quantity of pure mRNA-puromycin template could be eluted from the solid phase. Polyacrylamide gel analysis indicated a ligation yield of > 80 % for the mRNA-puromycin template which was directly used for translation and fusion formation in rabbit reticulocyte lysate to produce the mRNA-protein fusion library (7).We found the above protocol to be useful for efficient synthesis of mRNA-peptide and mRNA-protein fusion libraries from a number of template libraries different in length and complexity. Efficient mRNA affinity purification on neutravidin agarose carrying the biotinylated puromycin-linker replaced the laborious mRNA gel purification step used in previous protocols (3,5,7). Crosslink-formation of the hybridized mRNA on solid-phase and simultaneous biotin-tag cleavage yielded pure mRNA-puromycin template for direct use in an translation system. The ease with which the amount of synthesized mRNA-puromycin template can be controlled by hybridization with limiting amounts of linker allowed us to keep the reaction volumes and stoichiometry of the subsequent fusion synthesis constant and independent from the yield of the preceding transcription reaction. This feature of the present protocol should prove most useful in an automated protein evolution process which involves repetitive cycles of fusion library synthesis and selection and where manual quantification and adjustments only lead to a time-lag in the production process. Figure 2: Sequence design of the mRNA template 3’-end and the puromycin-linker. The mRNA template 3'-constant region codes for a linker hybridization sequence, a UAA stop codon and a dA18 tag sequence. The biotinylated puromycin-linker sequence shown in red carries a 5-terminal psoralen moiety (Pso), followed by a hybridization region and a flexible tether with puromycin (Pu) at the 3’-end and carrying a photo-cleavable biotin-tag. A 10 base complementary region between mRNA 3’-end and the photo-linker was found to be sufficient to achieve efficient hybridization and photo-crosslink formation. The use of 2'-OMe RNA in the linker hybridization sequence was intended to increase the stability of the 10 base-pair duplex; also, it protected the mRNA from cleavage by any RNase H activity during translation in rabbit reticulocyte lysate. The hydroxymethyl trioxsalen moiety (psoralen) was tethered to the 5'-phosphate through a hexamethylene spacer (8) (psoralen C6, Glen Research). METHODS: The puromycin-linker was prepared according to Kurz et al. (7) and biotinylated by carbamate bond formation between the puromycin amino group (50 µM) and the photo-cleavable biotin-reagent (9) (NHS-PC-Biotin, 5 mM, EZ-LinkTM-Biotin, Pierce Chemicals) in 25% DMSO/water for 2 h at room temperature followed by NaCl/EtOH precipitation. Photo-cleavage of the 1-(2-nitrophenyl)ethyl carbamate moiety restored the primary amino group on the 3’-terminal puromycin which acts as the peptidyl-acceptor in the fusion formation process (see Fig. 1).

Synthesis of milligram quantities of proteins using a ..

The originally described method for RNA-protein fusion preparation involved translation of an enzymatically linked mRNA-puromycin conjugate (3). Recently, we developed a simplified method for fusion synthesis from psoralen-crosslinked mRNA-puromycin templates (7). Here we report a further improvement of this method in which the mRNA-puromycin template is prepared in an integrated affinity purification and crosslink formation process on solid phase. This novel synthesis protocol further simplifies mRNA-protein fusion preparation and should prove most useful in an automated fusion synthesis process.