Los sistemas de producción en la zona afectada por la construcción

The original RNC production strategy is shown in Figure 3.3 A. To achieve the high concentration of ribosomes required for expression of RNCs, E. coli BL21 (DE3) are grown to a very high cell density (typically OD600=4 or higher) in MDG medium (based on an auto- induction medium [238]) for 16-20 h. It was found that growing the cells at 30 °C instead of 37 °C typically yielded a higher cell density, up to OD600=6 compared to ∼2. The end point OD600of the cells grown in MDG medium was typically used as an indication of cell viability, where cultures with OD600<2 were discarded.

After growth, the cells were harvested and transferred into expression medium that did not contain any carbon or nitrogen sources (Figure 3.3 A). The cells were incubated in this medium for a short period of time (∼25 mins) to allow depletion of all unlabelled carbon/nitrogen sources carried over from the MDG medium. At the end of the depletion period (indicated by a decrease in the OD600 reading, Figure 3.3 A), isotopically labelled carbon/nitrogen sources were added together with IPTG for expression.

Originally, minimal medium (M9) was used, which gave a yield of 100 pmol per litre. Repeated expression attempts did not result in an overall improvement in the yield, and therefore enhanced minimal media (EM9) was introduced as the expression medium. The level of RNCs expression was improved significantly, by an order of magnitude to >500 pmol per litre. At these expression levels, a band corresponding to the NC was easily observed by silver-stained SDS-PAGE. One minor problem was that the cells did not reach complete depletion as indicated by a drop in OD600for more than 1 hour, which is probably due to the rich components of the medium. The presence of any residual14N or12C during expression can lead to a decrease in NMR observable signals from the RNC. Instead, to rule out that the improved expression levels did not arise from a carry-over of nitrogen/carbon sources, a washing step (Figure 3.3 B) was introduced in place of the depletion stage to remove the residual unlabelled nitrogen/carbon sources from the expression medium.

In addition to the washing step, which enables 100 % labelling of the NC, rifampicin was added as a supplement to the EM9 medium to prevent background labelling of the ribosomes. Rifampicin is a bactericidal antibiotic that inhibits the natural bacterial RNA synthesis by inhibiting bacterial DNA-dependent RNA polymerase [239]. It binds to the RNA polymerase at a site next to the active centre and physically arrests RNA production [240, 241]. As the expression of the NC is under control of the T7 polymerase, it is not affected by rifampicin; adding rifampicin (150 mg l-1_{) to the expression medium after induction was found to reduce} the expression — and therefore labelling — of E. coli proteins, including ribosomal proteins, by ca. 50 % (Figure 3.4).

Time /hours OD 600 Growth in MDG no isotopes Expression in EM9 ₍15_N 13_C) 0 0 1 2 3 4 5 10 15 20 25 Change media, wash cells

B:

Cell growth/expression

A:

Cell growth/expression with depletion

OD 600 OD 600 Time /minutes Time /hours Harvest Harvest Growth Expression Expression Depletion Add 13_C/15_{N, IPTG}

Figure 3.3: A: The original growth and expression of RNCs using the depletion method at the expression stage. B: The optimised protocol for the growth and expression of RNCs to achieve unlabelled ribosomes and labelled NCs.

supplements (Cambridge Isotope Laboratories, Inc.) were also evaluated in the effort to further improve RNC expression levels. When added to EM9 medium, they had previously been found to improve the yield of ddFLN-RNCs, enabling short NC-length constructs (< 150 residues) to express at levels comparable to the full-length (220 amino acids) NC (Marilia Karyadi, personal communication). In the current study, the amino-acid supplements were used during the expression of wild-type (WT) α-synuclein (αSyn) RNCs, whose yield is typically five times lower than that of the ddFLN RNCs produced under the same expression conditions. The amino-acid supplements were found to improve the yield of the WT-αSyn RNC by three-fold (from 480 to 1500 pmol per litre, Annika Weise, personal communication).

The improvements that were developed during this study increased expression levels by ∼30 fold, and enabled RNCs suitable for RDC experiments to be produced.

B:

With Rifampicin 6.5 7.0 7.5 8.0 8.5 9.0 9.5 ppm δ_H

A:

Without Rifampicin 6.5 7.0 7.5 8.0 8.5 9.0 9.5 ppm δ_H 15N edited 15N filtered

Figure 3.4: Comparison of the amount of background labelling arising of ribosome in the absence (A) and presence (B) of rifampicin produced under RNC expression conditions. The

15_{N-edited spectra (red) contain signals solely from} 1_{H attached to} 15_{N and the} 15_N-filtered

spectra contain signals from1H not attached to15N. A: In the absence of rifampicin, the intensity of the 15N-edited spectrum is 24 % of the 15N-filtered spectrum, indicating 20 % labelling of the 70S N-H groups with15N. B: In the presence of rifampicin, the intensity of the15N-edited spectrum is 11 % of the15N-filtered spectrum, which means that 10 % of the 70S ribosome N-H groups are labelled with15N.

3.2.1.2 Development of the purification strategy for in vivo-derived RNCs

A critical step in RNC production is the separation of NC-occupied ribosomes from the released NCs, empty ribosomes and other endogenous proteins. During this work, the purification scheme described in [124] was extensively modified and improved.

There are two key requirements for RDC measurements of RNCs: that the samples are homogeneous, as impurities result in reduced sample lifetimes, and also that they are of high occupancy, i.e. that all the ribosomes contain a NC. The latter is especially important as the maximum working concentration of the ribosome is limited to 10 µM, which is a concentration that is at least an order of magnitude lower than that is typically used for NMR. A low occupancy therefore, will compromise the signals arising from the NC.

Illustrated in Figure 3.5 is the purification strategy used for in vivo RNCs, which was developed during the course of this study (in collaboration with Maria Karyadi, UCL). The strategy includes several key changes relative to the existing purification protocol described in [124], which were found to improve both the yield and purity of the RNCs and these are outlined below.

30 40 kDa 20

B:

Sucrose cushion

C:

Affinity chromatography

D:

Sucrose gradient

E:

Western blot detection

French press 4 passes 1000 Psi Spin cellular debris 45min 18,500 rpm

A:

Cell lysis Sucrose Cushion Sucrose Cushion 35% sucrose cushion 44000rpm 6-10hours Resuspend 70S pellet 60 - 80 nmol Released nascent chain Flow-through Load onto Ni-IDA beads

Elute Concentrate eluate through 100kDa cut-off concentrator 6 nmol 10% 35% 10-35% sucrose gradient 21000 rpm 15hours 1000pmol Ab 254 nm Fraction 10% 35%

kDa Concentrate Eluate through 100kDa cut-off concentrator

3 nmol Detection of NC and

accseement of occupancy

Figure 3.5: Schematic representation of a typical RNC purification following optimisation. A: Cell lysis with a French press preserves the ribosomal complexes. B: A sucrose cushion separates ribosomal material and large complexes from small soluble proteins. C: Metal- affinity chromatography with Ni-IDA resin separates nascent chain-occupied ribosomes from empty ribosomes. D: Sucrose gradient followed by fractionation separates intact occupied 70S ribosomes from 50S and 30S ribosomes and factors associated with the ribosome. The purified ribosome-containing fractions identified with silver-stained SDS-PAGE. E: A western blot against hexa-his tag assesses the occupancy and overall yield of the RNCs.

subjected to a sucrose cushion (Figure 3.5 B) (prior to metal affinity chromatography), which collects the ribosomal material within the pellet. The introduction of this step (optimised further as described in section 3.2.1.3) was found to improve the recovery of RNCs in the subsequent purification steps by enhancing the purity of the ribosomal material: specifically, the removal of released NCs and other endogenous E.coli proteins improved the binding of his-tagged RNCs to the metal affinity column (Figure 3.5 C). Using this approach, the overall yield of Dom5+110-RNCs improved two-fold to 100-200 pmol per litre.

separation of RNCs from empty ribosomes. The TALON resin (cobalt-affinity resin) was replaced with a Ni-IDA resin, as this was reported to have a higher binding capacity than TALON resin (200 mg/ml vs 5-18 mg/ml). The Ni-IDA resin was found to be highly selective for RNCs, and improved the recovery of Dom5+110-RNCs by a factor of 10. The use of the sucrose gradient as described in the original protocol was maintained as this was found to be essential in achieving highly homogenous samples as assessed by SDS-PAGE and western blot analysis (Figure 3.5 D&E). The buffer composition of the sucrose gradient was optimised to improve the overall yield of the RNCs; this is discussed in detail in section

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