- Research article
- Open Access
Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli
- Yi Liu†1,
- Wenjing Cui†1,
- Yueqin Fang2,
- Yuechun Yu1,
- Youtian Cui1,
- Yuanyuan Xia1,
- Michihiko Kobayashi3Email author and
- Zhemin Zhou1Email author
© Liu et al.; licensee BioMed Central Ltd. 2013
- Received: 30 January 2013
- Accepted: 30 May 2013
- Published: 3 June 2013
Activators of Nitrile hydratase (NHase) are essential for functional NHase biosynthesis. However, the activator P14K in P. putida is difficult to heterogeneously express, which retards the clarification of the mechanism of P14K involved in the maturation of NHase. Although a strep tag containing P14K (strep-P14K) was over-expressed, its low expression level and low stability affect the further analysis.
We successfully expressed P14K through genetic modifications according to N-end rule and analyzed the mechanism for its difficult expression. We found that mutation of the second N-terminal amino-acid of the protein from lysine to alanine or truncating the N-terminal 16 amino-acid sequence resulted in successful expression of P14K. Moreover, fusion of a pelB leader and strep tag together (pelB-strep-P14K) at the N-terminus increased P14K expression. In addition, the pelB-strep-P14K was more stable than the strep-P14K.
Our results are not only useful for clarification of the role of P14K involved in the NHase maturation, but also helpful for heterologous expression of other difficult expression proteins.
- N-end rule
- Pseudomonas putida
Nitrile hydratase (NHase, EC 126.96.36.199) is composed of α- and β-subunits. The enzyme contains either a non-heme iron (Fe-NHase)  or non-corrin cobalt ion (Co-NHase)  in the active center and catalyzes the hydration of a nitrile to the corresponding amide, which is followed by several consecutive reactions: amide → acid → acyl-CoA, as catalyzed by amidase  and acyl-CoA synthetase , respectively. The metal ions in both Co-NHase and Fe-NHase are located in their α-subunits, which share a characteristic metal-binding motif [CXLC(SO2H)SC(SOH)] containing two modified cysteine residues: cysteine-sulfinic acid (αCys-SO2H) and cysteine-sulfenic acid (αCys-SOH) [1, 4, 5]. The apoenzyme is likely to be unmodified, according to previous studies on NHase  and a related enzyme, thiocyanate hydrolase (SCNase) .
The trafficking of metal ions into NHases is mediated by various “activator proteins” . Fe-NHases require activators for functional expression in Rhodococcus sp. N-771 , Pseudomonas chlororaphis B23  and Rhodococcus sp. N-774 . A proposed metal-binding motif, CXCC, in the NHase activator of Rhodococcus sp. N-771 has been identified and the activators for Fe-type NHases have been shown to act as metallochaperones . For the two Co-NHases (L-NHase and H-NHase) in Rhodococcus rhodochrous J1, cobalt incorporation has been found to be dependent on self-subunit swapping: the activator protein exists as a complex with the α-subunit of NHase, the cobalt incorporation involves the swapping of the cobalt-free α-subunit of the cobalt-free NHase with the cobalt-containing α-subunit of the complex [13–15]. NHase in Pseudomonas putida NRRL-18668 and acetonitrile hydratase (ANHase, an NHase that catalyzes the hydration of small aliphatic nitriles) from Rhodococcus jostii RHA1 are also Co-NHases, in which P14K and AnhE, respectively, are essential for NHase maturation [16, 17]. However, their gene organizations are quite different from those of L-NHase and H-NHase. The structural genes of L-NHase and H-NHase have the order < β-subunit > <α-subunit > <self-subunit swapping chaperone>, while those in ANHase and the NHase of P. putida NRRL-18668 have the order < α-subunit > <AnhE > <β-subunit >  and < α-subunit > <β-subunit > <P14K > , respectively, with the latter protein being identical to the metallochaperone in Fe-NHase except that the molecular mass of the protein in Fe-NHase is larger than P14K. While AnhE has been found to act as a metallochaperone (not as a self-subunit swapping chaperone) during cobalt incorporation into ANHase , very recently, we discovered that cobalt incorporation into the NHase of P. putida NRRL-18668 is also dependent on the self-subunit swapping, and the P14K is a complex with the α-subunit . However, the P14K is difficult to be heterogeneously expressed, though a strep tag containing P14K was expressed, its low expression level and low stability retard the further clarification of their detailed role for cobalt incorporation.
In the present study, we successfully expressed the NHase activator P14K through site-specific mutagenesis taking into account N-end rule degradation. We also increased the expression and the stability of P14K by fusion of a pelB leader and strep tag together at its N-terminus. These results are useful for elucidation of the mechanism of cobalt incorporation into the α-subunit of NHase in P. putida NRRL-18668. Furthermore, these strategies, which promote the over-expression of the instable P14K in E. coli, might also be helpful for the heterologous expression of other difficult expression proteins.
Molecular modification to improve the yield of P14K from P. putidaNRRL-18668
Successful heterologous expression of P14K
N-terminal amino-acid sequence of NHase activators in various strains
N-terminal amino-acid sequence
Bordetella petrii DSM 12804
M K DERLPLP (YP_001630019.1)
Pseudomonas putida NRRL-18668
M K DERFPLP (P14K in this study*)
M K SCENQPN (AAF69003.1)
M K SCENQPN (AAS84452.1)
High P14K expression yield
Stability of pelB-strep-P14K
To investigate the mechanism of how the fusion protein pelB-strep enhances the recombinant expression of P14K, we compared the difference in the protein stability between the purified P14K-containing activator complex [α-(strep-P14K)] and [α-(pelB-strep-P14K)]. SDS-PAGE analysis was carried to investigate the stability of cobalt-containing [α-(strep-P14K)] and [α-(pelB-strep-P14K)] (a culture containing the cobalt ion) during storage at room temperature. As shown in Figure 5B, the pelB-strep-P14K band from the recombinant [α-(pelB-strep-P14K)] complex decayed to 90% of the original intensity after 2 days of storage and eventually to 60% after 6 days. However, the strep-P14K band from the [α-(strep-P14K)] complex decreased to 60% after only 1 day of storage and to 10% after 2 days. The finding that pelB-strep-P14K is far more stable than strep-P14K indicated that thermal stability may be a key factor in P14K expression.
In conclusion, the activator P14K from P. putida NRRL-18668 was successfully expressed based on the N-end rule degradation, the stability of the P14K was improved by adding a pelB signal peptide. Further study of the influence of P14K on the maturation of NHase is currently underway. The strategy used for P14K expression in this study may be useful for the heterologous expression of other difficult expression proteins.
Bacterial strain and plasmids
NHase and the P14K gene (ABP) were cloned from P. putida NRRL-18668. E. coli BL21 (DE3) was used as the host for the plasmid pET-24a(+), which was used for ABP, AB’P, PAB, ABPo, AB(strep-P), ABP(K2A), AB( △ N-P) and A(strep-P) expression. The plasmid pET-22b was used for (pelB-A)B(pelB-strep-P) expression.
Construction of plasmids
Oligonucleotide primers used in this study
Expression and purification of enzymes and enzyme assay
E. coli BL21 (DE3) transformants containing the recombinant plasmids were grown at 37°C in TB medium containing CoCl2.6H2O (0.05 g/l) and kanamycin (50 μg/ml) until culture A600 reached 0.8. Isopropyl β-D-thiogalactopyranoside was added to a final concentration of 0.4 mM. The cells were then incubated at 24°C for 16 h.
All purification steps were performed at 4°C. The procedures were conducted with an AKTA purifier (GE Healthcare UK Ltd.). Potassium phosphate buffer (KPB) (10 mM, pH 7.5) containing 0.5 mM dithiothreitol (DTT) was used in the purification steps. Both NHase and its activator complex were purified with a HisTrap HP column (GE Healthcare UK Ltd.). The target proteins were eluted off the column with gradient concentrations of imidazole from 0 mM to 500 mM (40 mM, 80 mM, 200 mM, 300 mM and 500 mM) in 10 mM KPB. The preliminarily separated proteins were further purified with a Hiload 16/60 Superdex 200 pg column (GE Healthcare UK Ltd.). The process of separation and purification was monitored by SDS-PAGE analysis.
NHase activity was assayed in a reaction mixture comprising 10 mM KPB (pH 7.5), 20 mM 3-cyanopyridine as a substrate and 0.1 μg enzyme in a total volume of 500 μL. The reaction mixture was incubated at 20°C for 20 min and terminated by addition of 500 μL of acetonitrile. The activity of NHase was determined by monitoring the formation of nicotinamide in the reaction mixture with high-pressure liquid chromatography (HPLC) as previously described . One unit of NHase activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of nicotinamide per min at 20°C.
This work was supported by the National Natural Science Foundation of China (31070711), the New Century Excellent Talents in University (NCET-10-0461), Fundamental Research Funds JUSRP20909 from the Central Universities 111 Project (111-2-06), the Doctoral Scientific Research Fund Project of Jiangnan University of China (JUDCF10011), the General University Doctor Research and Innovation Program of Jiangsu Province of China (CXZZ11_0475).
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