3. Results and Discussion
3.3. Protein Expression and Purification of the Recombinant Protein
For further optimization of the protein expression different protocols were tested: induction of the expression in an overnight culture for 2 to 6 hours, new culture inoculated with an overnight culture and expression at OD600 ~ 0.4 for 2 to 6 hours and induction of expression via culturing overnight. All attempts could not further increase the low protein expression level (fig 16A).
To test purification of the recombinant protein a 0.5 L culture (expression induced at OD600 ~ 0.4 for 4 hours with 1 mM IPTG) was lysed and incubated with Ni-NTA sepharose at 4 °C to bind the 6xHis-tagged protein to the matrix. After washing with buffer B (containing 20 mM imidazole) and elution with buffer C (containing 0.5 M imidazole) the purification steps were analyzed via SDS-PAGE. As shown in figure 16B only a small extent of the expressed protein bound to the Ni-NTA matrix and most of it could be found in the flow-through of the binding
3. Results and Discussion
Figure 16: SDS-PAGE analysis of (A) several protein expression protocols of polA*. M:protein ladder with molecular weight markers as indicated, 1-3:induction after growth over night, expression for 1:2 hours, 2:4 hours, 3:6 hours, 4-6 induction after growth up to OD600 0.5, induction for 4:2 hours, 5:4 hours, 6:6 hours. (B) Purification of polA* expression via Ni-NTA matrix. M:protein ladder with molecular weight markers as indicated, 1:cell lysate, 2:flow-through after binding, 3:first washing step, 4:second washing step, 5:elution fraction.
To optimise this purification procedure the buffer ingredients were varied and tested in different purification attempts to get better yields of polA* in the eluted fraction. Also other purification strategies via anion-exchange or heparin-sepharose or combinations of these two column systems did not result in detectable improvement of purification.
After that further E. coli BL21 strains were tested. First a BL21 pLysS strain was checked for better protein expression of polA*. Sometimes expression of special proteins is toxic for the expressing cell, which results in reduced growth and therby causes low levels of expressed protein. For this purpose pLysS strains were developed. They contain an additional plasmid in very low copy number, which encodes for T7 lysozyme. The expressed lysozyme will bind to T7 RNA polymerase and inhibits basal transcription before induction via IPTG. This will lead to better growth of the E. coli cells. After IPTG induction of protein expression overexpression of T7 RNA polymerase will render the inhibition by the small extent of lysozyme. Unfortunately expression in BL21 pLysS does not give higher expression levels of polA* (fig. 17B).
3. Results and Discussion
Then the BL21 Ril+ came into focus because they were developed to improve protein expression of non-bacterial proteins. Although D. radiodurans is a bacterium, the analysis of the protein and DNA composition of polA* (fig. 17A) shows a high percentage of used amino acid codons which are known to cause problems during recombinant expression in E. coli.
Normally bacteria prefer a special RNA codon of amino acids with redundant possible t-RNAs. Because of the high GC-content of the D. radiodurans genome, some of the used codons of the mostly affected amino acids (Arg - AGA, AGG; Ile - AUA; Leu - CUA; Pro - CCC; Gly) have a low abundance in E. coli. Forced high-level expression of a gene with codons that are rarely used by E. coli causes depletion of the internal t-RNA pools, called codon bias. Translation of the recombinant RNA is delayed, resulting in degraded proteins or codon substitutions and misincorporations that can destroy the functional characteristics of the protein. The RIL+ strain contains an additional plasmid which encodes supplementary t-RNAs for the amino acids Arg (argU), Ile (ileY) and Leu (leuW).
Figure 17: (A) Codon usage prediction of the first 300 amino acids of polA* in E. coli, whereas red dots stand for coding triplets which represent a bottle neck in protein biosynthesis [S3]. (B) SDS-PAGE analysis of polA*
expression in different E. coli strains.
While testing BL21 RIL+ for polA* expression one could observe increased expression rates (fig. 18B). The expression protocol was further optimised by analysing expression induction at different OD values and for different expression durations. As SDS-PAGE analysis (fig.
3. Results and Discussion
18A) shows, later induction of expression and longer incubation times could dramatically increase the yield of polA*. The expression condition no. 4, 6 and 14 (as indicated in fig.
18B) were choosen to perform purification of the expressed polA* protein with an ion-chelating affinity chromatography matrix (Ni-NTA batch, as described in the methods section). These results represent the successful purification of polA*. Expression in BL21 Ril+ leads to higher expression levels due to the suplementary t-RNAs and therefore a higher yield of the purified enzyme. Longer expression also increased impurity of the eluted polA*
fraction. For all further purifications expression was induced at OD600 ~0.5 for 4 hours at 37
°C.
Figure 18: SDS-PAGE analysis of (A) protein expression kinetics: N uninduced culture, 1-4:induction at OD600
~ 0.15, 5-8:induction at OD600 ~ 0.3, 9-12:induction at OD600 ~ 0.45, 13-16:induction at OD600 ~ 0.6; 1, 5, 9, 13:expression for 1 hour, 2, 6, 10, 14:expression for 2 hours, 3, 7, 11, 15:expression for 3 hours, 4, 8, 12, 16:expression for 4 hours. (B) Ni-NTA batch purification of polA* expression at conditions 4, 6 and 14 as described in A. F:flow-through after binding, w1-w3:washing steps 1 to 3, E:elution fraction.
To show that the purified enzyme is indeed polA* the SDS-PAGE (fig. 19A) containg all fraction of the purification procedure was blotted to a nitrocellulose membrane. Incubation with monoclonal anti-6xHis-antibodies, secondary anti-mouse-antibodies containing a horseraddish peroxidase and development of the fotofilm showed only bands at the expected size of 70 kDa (fig. 19B), verifying that the expressed and purified enzyme is polA*.
3. Results and Discussion
Figure 19: (A) SDS-PAGE analysis of polA* purification steps via Ni-NTA batch. M:molecular weight marker, L:lysate, F:flow-through, W1-W3:washing steps, E:elution fraction. (B) Western Blot of SDS-PAGE, index as described in A, incubated with primary monoclonal anti-6xHis antibodies and secondary anti-mouse antibodies.
(C) SDS-PAGE analysis of polA* expression in E. coli RIL+ and Rosetta strain and elution fraction of Ni-NTA purification.
A further increase of protein expression could be observed while using a BL21 Rosetta strain (fig. 19C) modified with an additional plasmid like RIL+, but with even more supplementary t-RNAs for the amino acids Arg, Ile, Leu, Pro and Gly.