Part:BBa_K4927057
HydABCC
HydABCC is a hydrogenase fused with the CL9 (BBa_K4927007) label on the N-terminal of the C subunit Dhydrogenase-C (BBa_K4927055). It will form an artificial multienzyme complex l in the experiment we designedTo improve the hydrogen production efficiency of the whole system.
Introduction
Hydrogenase is a metallic enzyme found in natural anaerobic bacteria (such as cyanobacteria) that catalyzes reversible reactions of hydrogen oxidation and reduction. According to the different metal elements contained in its active center, it can be divided into [Ni-Fe] hydrogenase and [Fe-Fe] hydrogenase. Among them, [Fe-Fe] hydrogenase is mainly used for proton reduction to produce hydrogen [86]. At present, four E. coli hydrogenase isoenzymes, hydrogenase 1 (Hyd-1), hydrogenase 2 (Hyd-2), hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4), have been reported to have hydrogen metabolism [87]. By analyzing the Syntrophomonas wolfei genome, Nathaniel et al. identified a polymeric [Fe-Fe] -hydrogenase (hyd1ABC), The gene sequence of this enzyme is similar to that of polymeric [Fe-Fe] -hydrogenase, NADPH-dependent [FeFe] -hydrogenase, and NADH-dependent formate dehydrogenase [88]. Polymeric [Fe-Fe] -hydrogenase can combine catalytic hydrogen oxidation and proton-reduction to produce hydrogen using equal molar amounts of NADH and reducing ferredoxin. This discovery has been confirmed in bacteria such as Thermotoga maritima and Acetobacterium woodii [89, 90]. They have also found that in the absence of ferredoxin, hyd1ABC may act as a non-polymerized, NADh-dependent [FeFe] -hydrogenase to reduce NADH to produce hydrogen. But maturation of [FeFe] -hydrogenase requires the involvement of a specific set of proteins (molecular chaperones) that synergically catalyze hydrogen biosynthesis [91]. Posewitz hydEF and found in the Rhine chlamydomonas hydG two genes, they mature in the biosynthesis of H clusters and enzymes in the process of coding [FeFe] - hydrogenase active protein needed to work [92]. Therefore, to synthesize biologically active mature [FeFe] -hydrogenase, co-expression of molecular chaperone (hydEFG) and hydrogenase (hyd1ABC) is required. Paul et al. cloned and expressed hydE, hydF and hydG homologues from Clostridium acetonbutanol, and established the biosynthesis system of [FeFe] -hydrogenase.
Usage and Biology
The NADH-dependent [Fe-Fe] hydrogenase (hyd1ABC) selected in this experiment was derived from Trophomonas wolfei and three hydrogenase subunits, namely hydA, hydB and hydC, were obtained by PCR reaction.We linked the C gene of poly [Fe-Fe] -hydrogenase (hyd1ABC) with CL9 gene, synthesized the hydrogenase C subunit sequence with CL9 tag, and connected A and B with CC fragment to construct the [Fe-Fe] -hydrogenase (HydABCC) plasmid; At the same time, we synthesized the chaperone vector (HydEFG). HydABCC was co-expressed with HydEFG gene, and mature [FeFe] -hydrogenase was obtained, thus the hydrogen production system was constructed. The system uses NADH as a substrate to catalyze hydrogen production without adding ferredoxin and other reaction additives.
Figure 1. HydABCC gene circuit
In addition, we used T5 exonuclease to routinely transform hydA, hydB and hydCC fragments into pETTrio vector skeleton to form Pettrio-CL9-hydabCC expression plasmid. In addition, gene fragments of hydE, hydF and hydG were fused with pRSFDuet vector skeleton through carrier ligations mediated by T5 exonuclide, and PRSFDUet-Hydefg chaperone promoted HydABCC expression vector was constructed.
Figure 2. Pettrio-CL9-HydABCC plasmid map.Note: M is protein ladder; The rest of the lanes are HydABCC eluted with different concentrations of imidazole.
We transferred the obtained expression vector into E.oil BL21(DE3) receptor cells, expanded culture for 8h, induced expression with 0.1mM IPTG for 16h, crushed the bacteria under low temperature and high pressure, and purified by Ni-NTA method to obtain recombinant HydABCC hydrogenase. The target protein was carried out on SDS-PAGE, and the final purification results were shown in Figure 1. The obtained HydABCC subunit sizes were consistent with the predicted protein molecular weights (hydA: 62.8 kDa, hydB: 44.8 kDa and hydCC: 31.9kDa).
Figure 3:HydABCC SDS-PAGE test result
Because [Fe-Fe] hydrogenase is triasylpolymerase, the correct expression of C subunit cannot be clearly seen in the SDS-PAGE diagram, and western blot assay (WB) should be performed on it. The subunits of [Fe-Fe] hydrogenase were connected by non-covalent bonds, and the 6×His label was placed on the N terminal of the CC subunit during vector construction in this experiment, so whether HydABCC was successfully expressed could be verified by WB results of the CC subunit. As shown in Figure 3.6, the correct expression of the [Fe-Fe] hydrogenase CC subunit (CC: 31.9kDa) indicates that we have successfully obtained the [Fe-Fe] hydrogenase.
Figure 4:Western blot results of HydABCC
In order to further obtain the high purity [Fe-Fe] hydrogenase, the purified hydrogenase was concentrated by ultrafiltration and purified by molecular sieve chromatography using AKTA protein purification instrument, and the high purity [Fe-Fe] hydrogenase was obtained. The protein standard and [Fe-Fe] hydrogenase were sequentially passed through the GE HiLoad 16/600 Superdex 200 prep grade chromatographic column, and the results were shown in Figure 3.7. Subsequently, the purified protein solution was subjected to SDS-PAGE electrophoresis, and the results were shown in Figure 3.8. According to the principle of molecular sieve chromatography, we can see that the molecular weight sizes of HydABCC subunits (A: 62.8 kDa, B: 44.8 kDa, C: 31.9kDa) were consistent with the predicted sizes. Compared with the standard protein, the results of hydrogenase chromatography showed that the second peak was the target protein, and the first peak might be the polysubunit oligomers (no active enzymes) produced when HydABCC was heteroglyphically expressed in Escherichia coli.
Figure 5. Molecular sieve chromatography results of HydABCC
Figure 6. Purification results of HydABCC. Note: M is protein ladder; The remaining lanes are HydABCC after nickel column purification and size- exclusion chromatography.
After obtaining hydrogenase, we used methylene blue REDOX titration to detect the activity of HydABCC by observing the change of color of the reaction system to determine whether hydrogen was generated. We took photos and recorded the color changes of the reaction at 0min, 5min and 10min (as shown in FIG.7). It can be seen that methylene blue gradually became colorless after the addition of hydrogenase, indicating the activity of HydABCC.
Figure 7. Results of methylene blue detection of HydABCC activity
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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