The Remarkable Yield Translation System (RYTS) Kit is a cell-free protein synthesis system. This kit includes an E.coli extract and all essential components for coupled transcription / translation reaction. The RYTS Kit contains the E.coli Lysate, which is prepared according to unique method developed for highly efficient production of extensive protein by RIKEN (1-4). ProteinExpress Co., Ltd. has been licensed to distribute this method by RIKEN under JP patent No. 4477923, US No. 7615344 and EP No.1655375.
Synthesis of recombinant protein using RYTS Kit is highly efficient, convenient and quick. Maximum yield of protein can reach 150μg per the 300μl reaction.
Furthermore, this kit is available for the expression of protein with unnatural amino acid by exploitation of CloverDirect™ tRNA Reagents for Site-Directed Protein Functionalization. CloverDirect™ allows the incorporation of unnatural amino acids at defined positions of proteins using in vitro translation (see page 8 for details). CloverDirect™ allows the incorporation of unnatural amino acids at defined positions of proteins using in vitro translation (see page 8 for details). If you need more information on CloverDirect™, please visit our web site.
<Evaluation of produced protein activity>
To evaluate performance of RYTS Kit in comparison to a competitor's cell-free protein synthesis kit (Kit A), activities of Green Fluorescent Protein (GFP) resulting from protein syntheses by these two kits were determined. After these reactions, the fluorescent intensities of each reaction mixtures were measured by a fluorescence scanner. It was shown that the obtained fluorescent intensity from RYTS reaction mixture was 3-fold greater than from Kit A reaction mixture
Figure 3. Comparison of produced GFP activity
Applied volume : 50 ml of translational reaction mix Fluorescence detection (Ex : 488nm / Em : 520 nm)
<Expression of site-directly labeled proteins>
A typical result of the protein labeling using RYTS and CloverDirect™ TAMRA (Product Code, #CLD02 and #CLD06) is show
Figure 4. In-gel detection of produced TAMRA labeled proteins
2UAG: UAG codon is inserted after initiator AUG codon
ProX tag*: ProX tag is fused to the N-terminus
Applied volume: 0.25 µl of RYTS translational reaction mix
White arrow: TAMRA labeled proteins
|1||white||E.coli Lysate||5 tubes||-80 °C|
|2||pink||2× Reaction Mix||5 tubes|
|4||orange||Enzyme Mix||1 tube||-20 °C|
|5||green||CAT Control Vector 250ng/ml||1 tube|
|6||clear||Nuclease Free Water||1 tube|
Construction and preparation of expression template
The DNA template (circular DNA or linear DNA) for protein synthesis should be contained that a protein-coding sequence be under the control of a T7 promoter and located downstream of a ribosomal binding site (RBS) sequence. Additionally, T7 terminator sequence is located at downstream of the coding sequence. Distance between T7 promoter and start ATG should not exceed 100 base pairs, and also distance between the RBS sequence and start ATG should not be more than 5-8 base pairs. Please refer to Figure 1 for an example of the DNA template.
Figure 1. An example of template
In case of very low protein yield, optimization of nucleotide sequence (codon usage, addition of N-terminal tags, etc.) is required to improve protein productivity. We recommend the use of ProX tag, which is original N-terminal peptide tag developed for efficient expression in E.coli cell-free protein synthesis system. ProX tag sequence is indicated at Figure 2.
|5’ ]||AUG||UCU||AAA||CAA||AUC||GAA||GUA||AAC||UUU||UCU||AAU||GAG||] 3’|
( B )
Figure 2. (A) ProX tag sequence, and (B) an example of template
< mRNA template>
Messenger RNA, which is generally synthesized from circular or linear DNA template using commercial transcription kit or RNA polymerase, is also available for RYTS reaction. In such case, the DNA template for transcription of mRNA should be also constructed according to section 5.1 DNA template. But, the T7 promoter, which is recognized by T7 RNA polymerase, can be replaced to other promoter recognized by other RNA polymerase (e.g., SP6 promoter recognized by SP6 RNA polymerase).
1) Cell-free protein synthesis system from Escherichia coli cells cultured at decreased temperatures improves productivity by decreasing DNA template degradation. Eiko Seki, Natuko Matsuda, Shigeyuki Yokoyama and Takanori Kigawa. Analytical Biochemistry, 377, 156-161 (2008)