Semisynthetic tRNA Complement Mediates <i>in Vitro</i> Protein Synthesis

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an <i>in vitro</i> protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative i<i>n vitro</i> peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly

Munc18c does not block SNARE assembly when the Sx4 C-terminus is immobilized.

<p>Coomassie Blue stained SDS-PAGE analysis of the binding of SNAP23 and VAMP2 to Sx4 proteins (Sx4_1-275-His or Sx4_1-275-T4L-His) immobilized on Co²⁺resin by their C-terminus. Immobilized Sx4 proteins were complexed with Munc18c (de-tagged) prior to overnight incubation with SNAP23 and VAMP2. C-terminally immobilized Sx4 proteins were able to pull down SNARE partners (SNAP23 and VAMP2) in the presence of Munc18c (lanes labeled Assembly) and in the absence of Munc18c (lanes labeled Controls). Lanes labeled INPUTS show the protein samples used in the experiments. The control lane labeled Munc18c is a negative control showing the lack of interaction of Munc18c with beads. The gel displayed is representative of three replicate experiments. Solid vertical lines on the gel image indicate the removal of intervening lanes or where two different gels have been placed adjacent to each other.</p

Munc18c prevents SNARE assembly when Sx4 C-terminus is not immobilized.

<p>Coomassie Blue stained SDS-PAGE analysis of the binding of SNAP23 and VAMP2 to Sx4s (de-tagged Sx4_1-275, Sx4_1-275-T4L or Sx4_1-298-TMD) bound to HL-Munc18c. HL-Munc18c was immobilized on Co²⁺resin and incubated for 6 h with Sx4_1-275, Sx4_1-275-T4L or Sx4_1-298-TMD and then allowed to interact with SNAP23 and VAMP2 overnight. The gel shows that SNAP23 and VAMP2 were pulled down by Munc18c:Sx4_1-275-T4L and Munc18c:Sx4_1-298-TMD but not by Munc18c:Sx4_1-275. The expected positions of pulled down SNAP23 and VAMP2 proteins are boxed. The gel displayed is representative of four replicate experiments. Lanes labeled INPUTS show the protein samples used in the experiments. Lanes labeled Controls, show that Sx4_1-275, Sx4_1-275-T4L or Sx4_1-298-TMD do not bind to the Co²⁺resin in the absence of bound HL-Munc18c.</p

Kinetics of Munc18c interaction with Sx4 in solution.

<p><b>a.</b> Time course of complex formation of Alexa488-labeled Sx4_1-275-His (50 nM) with HMunc18c at various concentrations: 100 nM (black curve), 200 nM (green curve), 300 nM (blue curve), 400 nM (pink curve), 1000 nM (red curve), 1500 nM (grey curve), 2000 nM (magenta curve) and 2500 nM (cyan curve). Solid lines represent global fits to titration data in the Dynafit 4.0 program. Resulting kinetic constants are shown in the graph inset <b>b</b>. As for <b>a</b>. except that formation of HMunc18c:Sx4_1-275-T4L-His was monitored. Concentrations of HMunc18c used were: 100 nM (black curve), 200 nM (green curve), 400 nM (blue curve), 600 nm (pink curve), 800 nM (red curve), 1000 nM (magenta curve), 1500 nM (gray curve).</p

Summary of kinetic data for the Munc18c-Syntaxin4 interaction.

<p>Values shown for the bio-layer interferometry experiments are mean ± s.d. where each experiment was repeated at least three times. Values and standard errors are shown for the fluorescence anisotropy experiments and these were obtained from a global fit to multiple kinetic data sets with differing concentrations of Munc18c. Bold text indicates dissociation rate constants (k_off) for Munc18c-Sx4_1-275 where Sx4_1-275 is free in solution.</p

Munc18c binds to Sx4 proteins immobilized by their C-terminus onto beads.

<p>Coomassie Blue stained SDS-PAGE gel showing that (after a 2 hr incubation at 4°C) de-tagged Munc18c is pulled down by all Sx4 proteins immobilized by their C-terminus, except Sx4_30-275-T4L which lacks the N-peptide sequence known to be important for Munc18c binding. There was negligible non-specific binding of de-tagged Munc18c to the metal affinity resin (lane labeled Bound, Munc18c). The expected position of Munc18c on the gel is boxed. The gel displayed is representative of three replicate experiments. Solid vertical lines on the gel image indicate the removal of intervening lanes.</p

Observed rate constants (k_obs, s^-1) of SNARE complex formation measured by fluorescence anisotropy (VAMP2 labeled) for Sx4 constructs in the presence and absence of Munc18c.

<p>Values are shown as mean k_obs ± s.d. from three independent experiments. Bold text indicates the observed rate constant (k_obs) for Sx4_1-275 SNARE assembly in the absence and presence of Munc18c showing that SNARE assembly is 7-fold slower in the presence of Munc18c for this construct only. Shading indicates all four experiments for which Munc18c was included, showing that Munc18c did not inhibit SNARE assembly when the Sx4 C-terminus had a T4L fusion, or when the Sx4 N-peptide was removed.</p

Munc18c reduces the rate of SNARE complex formation in solution when the Sx4 C-terminus is not anchored.

<p>Panels <b>a</b>. Sx4_1-275-His, <b>b.</b> Sx4_1-275-T4L-His, <b>c</b>. Sx4_30-275-His and <b>d.</b> Sx4_30-275-T4L-His show typical fluorescence anisotropy traces upon mixing SNARE complex components: 700 nM SNAP23, 100 nM Alexa488-labeled VAMP2 and 700 nM Sx4-variant alone (dark gray) or in the presence of 500 nM Munc18c (light gray).</p