Part:BBa_K1329005
NRPS: Amino acid activation domain
Idea
Our approach was to make it possible to combine the advantages of the nonribosomal peptide synthesis with the ribosomal pathway, i.e. the enormous repertoire of amino acids combined with the ability of the ribosome to synthesize huge proteins. To reach that aim, we planed to create a phusion protein that has the capability to activate amino acids derived from NRPSs´ A-domain PheA and tRNA binding capabilities derived from a part of multi aaRSs complexes Arc1p-C.
Cloning procedure
The PheA domain was amplified from the PheA-Arc1p-C-2x-pET28a construct and inserted into the linearized pET28a (XhoI, NdeI) cloning vector by Gibson assembly. To amplify the resulting plasmid Escherichia coli Top10 cells were transformed.
Test expression of PheA
After cloning procedures were successfully completed, a test expression of PheA was performed. Therefore E. coli BL21 (DE3) was transformed with the PheA plasmid. For the expression test, a 60 mL culture was grown to an OD of 0.5 and induced with 100 µM IPTG. Pre-induction (PI), induction (I) and two elution (E1, E2) fractions from the Ni-NTA were taken and prepared for SDS-PAGE analysis.
Gel analysis reveals that the transformed E. coli BL21 (DE3) strain produces the PheA on a small scale. The protein band appears at the expected size of 65 kDa so nevertheless an overexpression was conducted.
Production of PheA
Since the expression test was successful, protein expression was scaled up. For that purpose, an expression culture was inoculated and induced with 100 µM IPTG by an OD of 0.5.
Ni-NTA with anion exchanger and dialysis of PheA
Cells were harvested and the pellet was resuspended in buffer A before lysing the cells using a french press. After centrifugation the cleared supernatant (Load) was loaded on a column with Ni-NTA agarose by Qiagen. Gel analysis revealed that a band of the expected size is present in the elution fraction of the Ni-NTA. The eluted protein was concentrated and further purified using an anion exchanger. Possible fractions were analysed by SDS-PAGE.
Gel analysis revealed that a band of the expected size is present in the fraction 4 and 5. The eluted protein was concentrated and further purified by dialysis. The fractions were concentrated and stored at -80 °C until further use.
Measurement of aminoacylation levels
tRNA, amino acid and the fusion protein were incubated and the reaction started by the addition of ATP. The reaction was stopped by the addition of sodium acetate, tRNA was precipitated with ethanol and purified by size exclusion chromatography. Half of the sample was treated with base, reacidified and analysed by LCMS.
Measurements showed that all fusion constructs were able to load L- as well as D-phenylalanine onto tRNAPhe (Figure 2 a). The varying linker length showed a clear influence on the yield levels with the 2x-GSSG linker showing the highest catalytic activity yielding 11% loaded tRNA after 30 min while the 8x-construct reached only a maximum of 3% loaded tRNA as well as the linker-less version. The remaining constructs showed intermediate results. Furthermore the linking of the two domains leading to the increase in reactant concentration and the correct spatial arrangement is indeed important for the catalytic effect to occur since a mixture of the unlinked domains showed only background levels of aminoacylation (Figure 2 b). Further negative controls included testing the reaction without enzyme or ATP. As a positive control to evaluate the method phenylalanyl-tRNA synthetase (PheRS) was used. A time-dependent measurement of the aminoacylation level showed that a maximum is reached after 30 min (Figure 2 c). To test if other tRNAs except for the tRNAPhe can be aminoacylated using our fusion construct we carried out aminoacylation assays with five additional E. coli tRNAs (Figure 2 d). The measurements suggest in agreement with previous studies that all tRNAs were loaded similarly well.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 577
Illegal AgeI site found at 736
Illegal AgeI site found at 1228 - 1000COMPATIBLE WITH RFC[1000]
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