Difference between revisions of "Part:BBa K1329004"

Revision as of 16:31, 16 October 2014

NRPS: tRNA scaffold

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 planned to create a fusion 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 Arc1p-C 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 E. coli Top10 cells were transformed with it. For overexpression E. coli BL21 (DE3) was transformed with the resulting plasmid.

Test expression of Arc1p-C

After cloning procedures were successfully completed, a test expression of Arc1p-C was performed. Therefore E. coli BL21 (DE3) was transformed with the arc1p-C plasmid. For the expression test, a 60 mL culture was grown to an OD₆₀₀ of 0.5 and induced with 0.1 mM IPTG. Preinduction (PI), induction (I), pellet (P), supernatant (S), flow through (FT), wash (W) and elution (E1-3) samples were taken and prepared for SDS-PAGE analysis together with broad range marker (M) (New England Biolabs).

Gel analysis reveals that the transformed E. coli BL21 (DE3) strain produces Arc1p-C on a small scale. The purified protein shows the expected size of 22 kDa on a SDS-PAGE gel.

Production of Arc1p-C

Since the expression test was successful, protein expression was scaled up. For that purpose, an expression culture was inoculated and induced with IPTG (0.1 mM final concentration) when OD₆₀₀ reached 0.5.

Ni-NTA and anion exchange purification of Arc1p-C

Cells were harvested and the pellet was resuspended in buffer A before the cells were lysed using a french press. After centrifugation the cleared supernatant was loaded on a column with Ni-NTA Agarose (Qiagen). The eluted protein was concentrated and further purified using an anion exchanger. The combined fractions containing Arc1p-C were analysed by SDS-PAGE, concentrated and stored at -80 °C until further use.

Crystallization of Arc1p-C

Arc1p-C was crystallized using the sitting drop method in SWISSCI MRC 2 well crystallization plates with a drop size of 1 µL (1:1 mixture of protein and crystallization solution) with a reservoir volume of 50 µL. Crystals of Arc1p-C-single were obtained by room temperature and at 6 mg/mL protein concentration after 1 week in 0.1 M MES, pH 6.0, 10% (v/v) glycerol, 30% (w/v) PEG 600 and 5% (w/v) PEG 1000. To collect data the crystals were flash frozen in liquid nitrogen and measured at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France at beamline ID23-1. The structure of Arc1p-C-single was determined by molecular replacement (MR) using the crystal structure of human Arc1p-C (PDB ID: 1FL0).

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 (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 (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 (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 Escherichia coli tRNAs (d). The measurements suggest in agreement with previous studies that all tRNAs were loaded similarly well.

This domain originates from the aaRS accessory protein Arc1p from bakers yeast. It binds to a variety of tRNAs, and can be used as autonomous and transplantable module for tRNA recruitment into synthetic devices.

Sequence and Features