2D.1. Introduction
In view of the promising results obtained with the BACTH system concerning the positive interactions between TbAMPKα1/α2 and TbAMPKβ/γ and thus the identification of the catalytic subunits of the complex, it was decided to produce recombinant protein complexes of TbAMPKγβα1 and TbAMPKγβα2 using a prokaryotic polycistronic expression system.
Whereas several co-renaturation techniques of the complex from separately purified subunits have failed to reform the complex, and subunit expression alone has failed to produce soluble proteins, this technique has shown that it is possible to express the heterotrimeric complex. Originally developed by Neumann et al.[1], this technique aims at expressing several genes on a plasmid under the control of a single promoter in E.
coli cells. In addition, the presence of a His6 tag at the N-terminus of the TbAMPKα subunit will allow its isolation and purification. Compared to AMPK purified from cells or tissues, the complex produced by this method was homogeneous and did not contain kinase isoforms. Moreover, the fact that the complex purified with this method was fully functional, once activated, makes this expression system an interesting tool to study and characterize TbAMPK [1]. To date, all AMPK structural studies on mammals are based on this tri-cistronic expression system [2].
If all subunits of TbAMPK form a stable complex, then a polycistronix expression system is expected to provide recombinant heterotrimer. The successful expression and purification of a TbAMPK complex using these constructions will confirm the results obtained with the BACTH experiments (Part 2C), showing that both TbAMPKα1/α2 subunit form a complex with the regulatory subunits. Indeed, since the His6 tag necessary for the first purification step, nickel affinity chromatography, is on the TbAMPKα1/α2 subunit only if the catalytic subunit form a complex with the other subunit it will allow the purification of milligram amounts of TbAMPK as it is the case for mammalian AMPK [1]. This is very interesting when a large quantity of purified complex is required for further characterization.
Given that the three subunits are under the control of a single promoter, the expression level of the three subunits will be identical, which is important for efficient complex formation. If expression of all subunits in E. coli yields soluble proteins as is the case
for mammalian AMPK, this method will allow the production of both complexes TbAMPKγβα1/α2 suitable for further biochemical characterizations.
While subunits TbAMPKβ (Tb927.8.2450) and TbAMPKγ (Tb927.10.3700) are conserved, TbAMPKα still remains to be fully determined. Two candidates, TbAMPKα1 (Tb927.10.5310) and TbAMKα2 (Tb927.3.4560), of the catalytic subunit in TbAMPK and are investigated here. We therefore cloned the corresponding expression systems and we expressed and purified both heterotrimers.
2D.2. Materials and methods
2D.2.1. Generation of polycistronic expression vector
The creation of the polycistronic system was adapted from the previously created polycistronic expression system of mammalian AMPK from Neumann et al. in 2003 [1].
Whereas the global strategy was identical, the initial plasmid and the restriction enzymes used were different.
First the monocistronic plasmids harbouring either TbAMPKγ (55 kDa), TbAMPKβ (34 kDa), TbAMPKα1 (80 kDa) or TbAMPKα2 (70 kDa) (Figure 55 (A-C)) using pET series plasmids (pET30a and pET28b, Novagen) were cloned.
Figure 55 (A-C) The DNA sequence coding for the TbAMPK subunit were cloned into pET-plasmids in order to create monocistronic vectors. The insertion of the TbAMPKα1 and TbAMPKα2 subunit was in frame with the hexa-histidine tag present in the pET28b plasmid. The kanamycine resistance cassette (KanR) was present in the backbone of the plasmid.(D) General cloning strategy for polycistronic expression plasmid construction. The succession of restriction and ligation, starting from monocistronic plasmid carrying TbAMPKγ (55 kDa), TbAMPKβ (34 kDa), TbAMPKα1 (80 kDa) or TbAMPKα2 (70kDa) subunit respectively, in order to create the final plasmid were necessary. (E) Final sequence consisting of a promoter followed by the sequence of TbAMPKγ, TbAMPKβ, and His-TbAMPKα1/His-TbAMPKα2 all preceded by a ribosome binding sequence (RBS) and finally a transcriptional terminator. (Figure adapted from [1])
To this end, the TbAMPKγ gene sequence was amplified by PCR from gDNA of T.
brucei Nysm strain and integrated between NdeI and BamHI sites of a regular pET30a plasmid (pET30TbAMPKγ). This allowed the insertion of the gene of interest just after the Ribosome Binding Site (RBS) sequence. The same protocol was used to obtain a plasmid harbouring TbAMPKβ (pET30TbAMPKβ). The gene sequences
corresponding to the TbAMPKα subunits (TbAMPKα1 and TbAMPKα2) were amplified and subsequently inserted into pET28b between the NdeI and SalI sites, affording pET28TbAMPKα1 and pET28TbAMPKα2, in frame with the His6 tag provided by the MCS of pET28b (His6-tag located at the N-terminus of TbAMPKα1/α2 subunits).
Furthermore, the presence of a thrombin recognition sequence between the Hi6-tag in N-terminus and the beginning of the gene will allow to cleave the tag if needed for further experiments.
Reverse primers for all subunits were designed to contain a SpeI restriction site upstream and adjacent to the terminal restriction site used for cloning. All primers used for the cloning process are presented in Table 21.
Two repeating steps of digestion and ligation were necessary to create the final tricistronic expression system. First, pET30TbAMPKβ was digested with XbaI/XhoI and the fragment, containing the RBS and the gene, were ligated into pET30TbAMPKγ digested with SpeI/XhoI (XbaI and SpeI digestion creates two compatible cohesive ends that, upon ligation, do not recreate a XbaI or SpeI recognition site), yielding pET30TbAMPKγβ. Then, pET28TbAMPKα1 and pET28TbAMPKα2 were digested with XbaI/XhoI, and the corresponding fragments, containing a RBS sequence and the His6 tag before the gene, were ligated into pET30TbAMPKγβ previously digested with SpeI/XhoI, providing pET30TbAMPKγβα1-His and pET30TbAMPKγβα2-His, respectively. The schematic construct is showed in Figure 55 (D-E) and the sequence is shown in Appendix B. Table 22 shows the final clones obtained and used for the experiments.
Table 21 Primers used for cloning of the polycistronic expression system.
Primers Sequence* PCR condition
and DNA source
Table 22 Final clones used for the polycistronic experiment
plasmid Final clone
Both expression constructs were verified by automated sequencing (Microsynth AG, Switzerland). The presence of the His6 tag at the N-terminus of either TbAMPKα subunits allows the purification of the protein (or the complex) using metal chelate affinity chromatography. The presence of the RBSs assure the recognition of the sequence by the ribosomes that will continue the expression of the proteins instead of stopping the translation after the first stop codon of the TbAMPKγ subunit.
2D.2.2. Protein complex production
The final plasmids were transformed initially into BL21-Codon Plus(DE3)-RIL and later into LOBSTR-BL21(DE3)-pLysSRARE. Starting from a glycerol stock a 10 mL preculture was grown in 2xTY medium during the day in presence of kanamycine (50 µg/mL) and chloramphenicol (34 µg/mL) at 37°C. 1 L of Terrific Broth (Difco) was inoculate with 1 mL of preculture (in presence of antibiotics) and incubated over night at 37°C while shaking at 150 rpm. The culture was cooled down to 12°C before protein production was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 72 h. Bacteria were harvested by centrifugation (15 min at 5'000 rpm) and the pellet was stored at -20°C. Other induction conditions were tested: 24h induction at 25°C and 4-5h induction at 37°C.
2D.2.3. Protein complex purification
Nickel affinity chromatography: Cells were suspended in 30 mL of lysis buffer (20mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 10 mM ATP, 10 mM MgCl2, 2 mM β-mercaptoethanol, pH 8) containing DNAse and protease inhibitor cocktail (Sigma) and lysed twice with a French Press. The supernatant obtained after centrifugation at 15’000 rpm (40 min, 4°C) was filtered and manually loaded on the HiTrap Metal chelate column (GE Healthcare Life Science) charged with Nickel. The column was then connected to the AKTA FPLC (Amersham Biosciences) and after washing with lysis buffer, bound proteins were eluted with a linear imidazole gradient using elution buffer (20mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, 10 mM MgCl2, 2 mM β-mercaptoethanol, pH 8) at a flow rate of 2 mL/min (20 column volumes (CV)). Fractions of 2 mL were collected and analysed by 12% SDS PAGE.
Anion exchange chromatography: Fractions containing the heterotrimeric complex from Nickel affinity chromatography were pooled and diluted 4 times with buffer A (20 mM Tris-HCl, 1 mM DTT, pH 8) in order to reduce the salt concentration in the sample on a HiTrap Q XL column (Amersham Bioscience). Then the solution was loaded and equilibrated with12% of buffer B (20 mM Tris-HCl, 1 M NaCl, 1 mM DTT, pH 8) in A.
Protein was eluted with linear gradient starting from 12% buffer B at a flow rate of 2 mL/min (12 CV). Fractions of 2 mL were collected and analysed by 12% SDS PAGE.
Size Exclusion Chromatography (SEC): Fractions of interest from anion exchange chromatography were pooled and concentrated using Spin-X UF columns (50 kDa cut-off, Corning). Purified TbAMPK was 0.45µM filtered and injected by means of a 1 mL loop and loaded to a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Science) equilibrated with buffer C (20mM Hepes, 150mM NaCl, 1 mM TCEP, pH 7.4) or PBS (8 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 1 mM TCEP, pH 7.4) depending on the experiment performed. The flow rate was set at 0.75 mL/min, and fractions of 0.5 mL were collected and analysed by 12% SDS PAGE.
Thrombin digestion experiments: Thrombin recognizes the sequence Leu-Val-Pro-Arg-Gly-Ser and cleaves between Arg and Gly. In this case, it is used to cleave the His6-tag attached to the subunit TbAMPKα2. Table 23 presents the volumes used for the thrombin digestion. The solutions were incubated at 37°C and aliquots of 15 μL were taken right after addition of the thrombin and every 15 min until 1 h. The sample were analysed by SDS PAGE. Control samples were taken at the beginning and end of the experiment.
Table 23 Solution volumes for thrombin digestion and control
Trombin digestion Control TbAMPK
Trombin 1U/μL 5 μL /
CaCl2 100mM 2 μL 1 μL
Purified TbAMPK
193 μL 96.5 μL
ddH2O / 2.5 μL
2D.2.4. Western Blot
The Western Blot analysis was performed as previously described (Part 2A, 2.2.8.).
The first antibody in blocking solution was incubated o/n at 4°C on a rocking platform and after several washes, the secondary antibody was incubated for 90 min again at RT on a rocking platform. Table 24 present the antibodies (primary and secondary) used in this work diluted in 3%BSA in TBS-T.
Table 24 List of primary and secondary antibodies.
Tag/epitope 1st antibody 2nd antibody Detection system His-tag mouse anti-His,
The creation of a polycistronic prokaryotic expression system containing the three TbAMPK subunits under the control of a single promoter was achieved. Several cloning steps were necessary to create the two polycistronic expression systems TbAMPKγβ-Hisα1 and TbAMPKγβ-Hisα2 respectively. First, each gene was integrated into a plasmid of the pET series to produce monocistronic plasmids carrying the gene in-frame with the ribosome binding site (RBS) sequence. Second, the subsequent digestion and ligation steps allowed cloning of all subunits into the monocistronic plasmid carrying the TbAMPKγ subunit. The final plasmid construct constituted of the three gene sequences only separated by a 50 bp long intergenic sequence harbouring the RBS and with the TbAMPKα subunit being in frame with a N-terminal His6-tag (pET30TbAMPKγβHis-α1 or pET30TbAMPKγβHis-α2). The His6-tag present allowed the purification by Nickel affinity chromatography of the complex expressed and formed in E. coli (BL21-(DE3) or LOBSTR-BL21(DE3)-RIL-pLysSRARE [3]). The tag is present only on the catalytic subunit. Thus in nickel affinity purification only candidates that form a stable complex with the regulatory subunits will be co-purified.
It was decided to use a particular E. coli strain, LOBSTR-BL21(DE3) for all subsequent expression. This strain was developed to reduce co-purification of naturally occurring histidine-rich proteins in bacteria that are well known to contaminate eluates from Nickel Affinity Chromatography purifications [3]. In the LOBSTR strain, two proteins, ArnA (which contains several histidine residues on the surface) and SlyD (which has a long C-terminal tail rich in histidine residues), have been genomically modified to reduce their affinity for nickel [3].
For protein production, several conditions were tested for both constructs: 12°C for 72 hours, 25°C for 24 hours and 37°C for 3-4 hours.
2D.3.2. Isolation and purification of TbAMPKγβα1
After induction with IPTG and protein production at 25°C and 12°C the clarified bacterial lysate was analysed by Nickel affinity chromatography. The purification of the protein obtained after expression in bacteria at 25°C and 12°C shows that only small signals are observable (Figure 56 (A and C)). Subsequent SDS-PAGE analysis of the crude extract, the soluble/insoluble fraction as well as the eluted fractions did not show the proteins of interest and of the expected size, i.e. TbAMPKγ (55kDa), TbAMPKβ (35kDa) and TbAMPKα1 (80kDa) (Figure 56 (B and D)). Some faint bands around 80 kDa could be observed, but western blot analysis of these fractions using an anti-His antibody showed that they did not correspond to the TbAMPKα1 subunit (Figure 57).
Figure 56 Chromatogram of TbAMPKγβHis-α1 produced at 25°C (A) and 12°C (C) after Nickel-affinity chromatography with HiTrap column. Coomassie stained SDS-12%PAGE analysis of fraction of interest of TbAMPKγβHis-α1 produced at 25°C (B) and 12°C (D).
The bands observed in the coomassie stained gel most likely correspond to unspecifically bound proteins, for example heat shock proteins that often co-purify at low temperature expression and when difficult protein are expressed [4].
The results of the purification suggest that subunit TbAMPKα1 may not be an acceptable candidate for TbAMPKβ and TbAMPKγ to form a stable complex, thus not allowing the isolation of the heterotrimeric complex. However, this is in contrast with the findings obtained with the BACTH analysis (Part 2C) which clearly identified TbAMPKα1 as a functional α1 subunit for TbAMPK.
Western blot analysis clearly shows that TbAMPKα1 is not expressed thus no complex is present either because the subunit is not soluble, form inclusion bodies, or because it is not stable and is quickly degraded in bacteria. Moreover, the presence of the tag,
even if it is very small, can destabilize interactions as it was demonstrated in the BACTH experiment with N-terminally labelled TbAMPKα subunits using larger tags.
Figure 57 Western Blot analysis of Nickel affinity chromatography fractions 6 and 7 of TbAMPKγβα1 produced at 12°C. Mouse antibody anti-His 1:2500 and goat antibody anti-mouse IgG 1:25000. Positive control: sample of total protein extraction of insect cells containing a 130 kDa His-tagged protein.
2D.3.3. Isolation and purification of TbAMPKγβα2
As for the TbAMPKγβ-Hisα1, plasmid pET30-TbAMPKγβ-Hisα2 was successfully cloned and apression was carried out at 37°C, 25°C and 12°C (Figure 58-Figure 60).
The purification of the complex expressed at 37°C showed similar results as with TbAMPKα1. Only a small peak could be observed in the chromatogram of the Nickel-affinity chromatography and only very weak signals were detected in the Coomassie-stained gel loaded with the fractions of interest. Further SEC analysis, after the concentration of the collected fraction, did not allow the detection of the complex (Figure 58 (D)).
Figure 58 (A) Chromatogram of TbAMPKγβα2 produced at 37°C in BL21(DE3)-RIL E. coli after Nickel affinity chromatography. (B) Coomassie stained SDS-12%PAGE analysis of fraction of interest of TbAMPKγβα2. (C) Chromatogram of SEC (fractions 6-8 of Nickel affinity chromatography). (D) Coomassie stained SDS-12%PAGE analysis of fractions 18-20 corresponding to the TbAMPK complex and others fraction corresponding to the others peaks observed.
Interestingly, the complex expressed at 25°C showed a slightly bigger peak (at 12.19 mL elution volume) compared to the one observed before (Figure 59 (A)). The corresponding Coomassie-stained gel loaded with the fractions of interest (fractions 6-9) showed at least three proteins that could correspond to TbAMPKα2 (70 kDa), TbAMPKβ (35 kDa) and TbAMPKγ (55 kDa) (Figure 59(B)).
Figure 59 (A) Chromatogram of TbAMPKγβα2 produced at 25°C in BL21(DE3)-RIL E. Coli after Nickel affinity chromatography. (B) Coomassie stained SDS-12%PAGE analysis of fractions of interest of TbAMPKγβα2. (C) Chromatogram of SEC (fractions 5-9 from Nickel affinity chromatography). (D) Coomassie stained SDS-12%PAGE analysis of fractions 19-23 corresponding to the TbAMPK complex and others fraction corresponding to the others peaks observed.
The fractions containing the complex of interest (5-9) were pooled, concentrated, filtered and analysed by SEC, and the SDS-PAGE showed three very well defined bands of the expected size in fractions 19-23 (Figure 59 (D)).
The best results were obtained with an expression temperature at 12°C (Figure 60).
The TbAMPKγβHis-α2 complex is clearly visible in fractions 5-9 (Figure 60 (A-B)).
Further gel filtration could remove most of the contaminants, yielding three pure bands, that are likely to correspond to TbAMPKα2 (70kDa), TbAMPKβ (35kDa) and TbAMPKγ (55kDa) in fractions 19-23 (Figure 60 (D)).
Figure 60 (A) Chromatogram of TbAMPKγβα2 produced at 12°C in BL21(DE3)-RIL E. Coli after Nickel affinity chromatography. (B) Coomassie stained SDS-12%PAGE analysis of fractions of interest of TbAMPKγβα2. (C) Chromatogram of SEC (fractions 5-9 of Nickel affinity chromatography). (D) Coomassie stained SDS-12%PAGE analysis of fractions 19-23 corresponding to the TbAMPKγβα2 complex and others fraction corresponding to the others peaks observed.
As a next step, the presence of the TbAMPKα2 subunit was confirmed by western blot probing against the His-tag. Both the crude extract and the SEC fraction 21 (from purification at 12°C Figure 60) showed a strong signal at 70kDa, thus the protein observed corresponds, indeed, to TbAMPKα2 subunit (Figure 61). This shows that in contrast to candidate TbAMPKα1, the TbAMPKα2 subunit is soluble and able to form a strong complex with the regulatory subunits, being able to withstand bacterial lysis as well as all purification steps. This observation goes against previous conclusions (Parts 2A and 2B), that stated that the impossibility to obtain the heterotrimeric complex was eventually due to its instability during purifications.
Figure 61 Western blot analysis of TbAMPKγβα2 Nickel-affinity crude extract and SEC fraction 21 using mouse antibody anti-His 1:2500. Positive control: sample of total protein extraction of insect cells containing a 130 kDa His-taged protein.
Comparison of the production of TbAMPKγβHis-α2 at different temperatures
The comparison of the purification of TbAMPKγβHis-α2 produced at different temperatures was carried out. Having difficulty measuring the protein concentration of the final elution in these preliminary tests, the comparison was based on the amount of TbAMPKγβHis-α2 produced from an equal amount of initial E. coli BL21-Codon Plus(DE3)-RIL cells. As production at 37°C was the lowest, the amount to be loaded was based on the maximum production after SEC analysis at 37°C. Based on these preliminary tests, it was clear that the best yield was obtained with production at 12°C for 72 hours (Figure 62). For this reason, all subsequent purifications were carried out under these conditions.
Figure 62 Comparison of the TbAMPKγβα2 produced at different temperatures. (A) Table summarizing the amount of final eluate charged on the gel. (B) Coomassie-stained SDS-12% PAGE gel charged with equal amount of initial bacterial wet pellet.
2D.3.4. Expression of the TbAMPKα2 subunit
After the successful purification of the complete complex, the investigation of the TbAMPKα2 subunit alone was carried out to confirm that in order to obtain a stable and active complex all subunits need to be expressed together. On the one hand, the purification of a stable and complete TbAMPKα2 subunit alone would be very interesting for further crystallography experiments, investigations of the activation loop and activity experiments. On the other hand, if active alone, it will indicate that TbAMPKα2 is a kinase itself and that the presence of the regulatory subunit to form the heterotrimeric complex is redundant. pET28TbAMPKα2 was transformed into LOBSTR-BL21(DE3) and protein expression was performed at 12°C. The fraction of interest after Nickel-affinity chromatography (chromatogram shown in appendix C) were analysed by SDS-PAGE (Figure 63 (A)). A series of protein bands can be distinguished, a feint band at 70 kDa, and three major bands at 60 kDa and between 35-40 kDa (fractions 6-10). These bands are not present in the same fractions of the purification of the heterotrimeric complex TbAMPKγβHis-α2 (Figure 60, Figure 62 or Figure 65).
In order to determine the origin of the appearance of these three intense bands, a Western blot against the His6 tag was carried out (Figure 63 (B)). In the blot, in addition
to the detection of a 70kDa band and a 60 kDa band, a strong signal is detected between 35-40kDa, corresponding to the bands observed in the Coomassie stained gel. These observations suggest that these bands correspond to different C-terminal truncated versions of TbAMPKα2 subunit as they still carry the N-terminal His6-tag.
Apparently the TbAMPKα2 subunit could be expressed as soluble protein, but lacks proteolytic stability when expressed alone. Since the purification was carried out in presence of protease inhibitors, this degradation already occurred during expression in bacteria.
Figure 63 (A) Coomassie-stained SDS-12% PAGE analysis of Nickel-affinity chromatography fractions for TbAMPKα2. (B) Western Blot using mouse antibody anti-His 1 :2500.
As an alternative method, the proteins in fractions 6-10 were further analysed by thrombin digestion. Thrombin is a serine protease enzyme that recognizes the sequence composed of Leu-Val-Pro-Arg-Gly-Ser that is located between the
As an alternative method, the proteins in fractions 6-10 were further analysed by thrombin digestion. Thrombin is a serine protease enzyme that recognizes the sequence composed of Leu-Val-Pro-Arg-Gly-Ser that is located between the