Skip to main content
Download PDF

Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli

BMC Biotechnology volume 13, Article number: 48 (2013) Cite this article

Abstract

Background

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.

Results

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.

Conclusions

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.

Background

Nitrile hydratase (NHase, EC 4.2.1.84) is composed of α- and β-subunits. The enzyme contains either a non-heme iron (Fe-NHase) [1] or non-corrin cobalt ion (Co-NHase) [2] 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 [3] and acyl-CoA synthetase [1], 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 [6] and a related enzyme, thiocyanate hydrolase (SCNase) [7].

The trafficking of metal ions into NHases is mediated by various “activator proteins” [8]. Fe-NHases require activators for functional expression in Rhodococcus sp. N-771 [9], Pseudomonas chlororaphis B23 [10] and Rhodococcus sp. N-774 [11]. 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 [12]. 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 > [16] and < α-subunit > <β-subunit > <P14K > [17], 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 [16], 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 [18]. 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.

Heterologous expression systems are commonly used for protein research. Protein degradation in heterologous expression systems often leads to failure for the isolation of proteins of interest. Intracellular protein degradation plays an essential role in many physiological processes by removing damaged polypeptides and proteins that harbor specific destruction tags. N-end pathway degradation relates the metabolic stability of a protein to the N-terminal residue of that protein [19]. The N-end rule defines the stability of proteins according to the nature of their N-terminal residues. These residues are classified as stabilizing and destabilizing residues, which serve as recognition determinants for protein degradation [19–21]. In bacteria such as E. coli, the N-end rule pathway is present. According to the N-end rule, amino-terminal arginine, lysine, leucine, phenylalanine, tyrosine and tryptophan confer 2 minute half-lives to proteins, while the other amino-terminal residues confer greater than 10 hour half-lives to the same proteins (Figure 1) [22, 23].

Figure 1
figure 1

The schematic diagram of N-end rule in bacteria. (A) Relatively long protein half-life. If the second N-terminal amino-acid is A, G, S, C or P and in some cases V or T, which can be processed by methionine aminopeptidase (MetAP), the protein will have a long half-life. (B) Relatively short protein half-life. When the second N-terminal amino-acid is W, Y, F, L, R, K, M, D, E, I, H, Q or N, which can not be processed by MetAP, the protein will have a short half-life.

Full size image

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.

Results and discussion

Molecular modification to improve the yield of P14K from P. putidaNRRL-18668

To clarify the mechanism of cobalt incorporation into the NHase of P. putida NRRL-18668, we attempted to isolate P14K, which is essential for NHase activity [17]. The transformant harboring pET-ABP, which contained the NHase and P14K genes, was used for NHase and P14K expression. As a result, although NHase was successfully expressed, P14K was hardly detected on SDS-PAGE. This result is consistent with the previous report [17]. Although a strep tag containing P14K was expressed using a transformant harboring pET-AB(strep-P), and the strep-P14K was found to be a complex with the α-subunit [α-(strep-P14K)] [18], the low expression level and the low stability of the complex retard the further clarification of its detailed role for cobalt incorporation, a strategy for enhancing P14K expression level and stability is expected. It has been shown that heterologous expression of proteins in E. coli can be enhanced by using a synthetically strong ribosome binding site (RBS) [24], reducing the distance between the promoter region and target gene [25] and gene codon optimization [26]. To enhance the expression of P14K, the putative RBS (CTGGAG) within the B gene (encoding the β subunit) (Figure 2A) was replaced with an enhanced RBS (AAGGAG) between gene B and gene P (encoding P14K) during the construction of the plasmid pET-AB’P (Figure 3). Simultaneously, the plasmid pET-PAB, with the gene order < P > <A > <B>, was constructed. In this plasmid, the distance between the promoter sequence and the P14K gene was shortened and the strong RBS was inserted upstream of each gene (Figure 3). Moreover, plasmid pET-ABPo, with optimized P14K gene codons and a strong RBS for each gene, was also constructed (Figure 2B). However, P14K was not abundantly expressed by the transformants harboring pET-AB’P, pET-PAB or pET-ABPo (Figure 4A).

Figure 2
figure 2

Potential ribosome-binding site of P14K and the codon-optimized nucleotide sequence of P14K. (A) Nucleotide sequence of P14K. The underlined nucleotide sequence denotes the potential ribosome-binding site. (B) Nucleotide sequence of the codon-optimized P14K (Po) nucleotide sequence.

Full size image
Figure 3
figure 3

Sketch of the plasmids used in this study. pET-ABP, a plasmid containing the ABP genes; pET-AB’P, a plasmid containing the ABP genes with an enhanced RBS (AAGGAG) between the B and P genes; pET-PAB, a plasmid containing the ABP genes with a < P > <A > <B > order; pET-ABPo, a plasmid containing the AB and the codon-optimized P genes; pET-AB(strep-P), a plasmid containing the AB and a strep-tagged P genes [18]; pET-ABP(K2A), a plasmid harboring the AB and a mutant P (the second lysine (K) is mutated to alanine (A)) genes; pET-AB(△N-P), a plasmid harboring the AB and a 16 amino-acid truncated N terminus P genes; pET-A(strep-P), a plasmid containing the A and a strep-tagged P genes; pET-(pelB-A)B(pelB-strep-P), a pET-22b plasmid containing the pelB-A, B and pelB-strep-P genes.

Full size image
Figure 4
figure 4

SDS-PAGE analysis of recombinant NHases and P14K, and the predicted secondary structure of P14K. (A) SDS-PAGE analysis of the cell extracts of the transformants carrying pET-ABP, pET-AB’P, pET-PAB or pET-ABPo. 1, mark; 2, pET-ABP; 3, pET-AB’P; 4, pET-PAB; 5, pET-ABPo. (B) Predicted secondary structure of P14K. (C) SDS-PAGE analysis of the cell extracts of the transformants carrying pET-AB(strep-P), pET-ABP(K2A) or pET-AB(△N-P). 1, mark; 2, pET-ABP; 3, pET-AB(strep-P); 4, pET-ABP(K2A); 5, harboring pET-AB(△N-P).

Full size image

Successful heterologous expression of P14K

The N-end rule states that the half-life of a protein is determined by the nature of its N-terminal residue [19–21]. This fundamental principle of proteolytic regulation is conserved from bacteria to mammals [19]. N-terminal arginine, lysine, leucine, phenylalanine, tyrosine and tryptophan confer 2 minute half-lives to proteins, while the other N-terminal residues confer greater than 10 hour half-lives to the same proteins [22, 23] (Figure 1). We analyzed the N-terminal amino-acids of some weakly expressed NHase activators from Bordetella petrii DSM 12804, P. putida NRRL-18668, Bacillus BR449 and Bacillus RAPc8. Surprisingly, we found that all of the second N-terminal amino-acids of the NHase activators in these four strains are lysine (K) (Table 1). Thus, it may be that N-end rule degradation leads to difficulty with the expression and isolation of P14K. Based on this speculation, we designed a mutant gene, ABP(K2A), in which the Lys-2 in the N-terminus of P14K was substituted with Ala in the plasmid pET-ABP(K2A). In addition, we analyzed the secondary structure of the P14K as predicted by JPRED3 (http://www.compbio.dundee.ac.uk/www-jpred/index.html) and found the N-terminal 16 amino-acid group is in an N-terminal loop (Figure 4B) and the 17th amino-acid is a Gly that could avoid P14K degradation effectively according the N-end rule [22, 23]. Therefore, we constructed plasmid pET-AB(△N-P), in which the N-terminal 16 residues of P14K were deleted, the second N-terminal amino-acid in the truncated P14K was Gly. The transformants harboring pET-ABP(K2A) and pET-AB(△N-P) were used for NHase and P14K expression. As shown in Figure 4C, each mutant P14K was successfully expressed and the crude NHase activity in the cell-free extracts of each transformant was similar to that of the transformant harboring pET-ABP (120.5 U/mg). However, compared with the expression of P14K using the transformant harboring pET-AB(strep-P), the expression level of P14K in the two mutants was not improved (Figure 4C).

Table 1 N-terminal amino-acid sequence of NHase activators in various strains
Full size table

High P14K expression yield

It has been reported that exporting a protein to the periplasm to enhance its stability can be regarded as an effective strategy to optimize the production of recombinant proteins [27, 28]. To yield large amounts of P14K in recombinant E. coli cells, we attempted to secrete P14K into the periplasmic space. As P14K formed a complex with the α-subunit of NHase [18], we designed a mutant gene (pelB-A)B(pelB-strep-P) in which the pelB signal peptide was added upstream of the A and strep-P genes in the plasmid pET22b-(pelB-A)B(pelB-strep-P). The transformant harboring pET22b-(pelB-A)B(pelB-strep-P) was used for recombinant P14K expression. Although no target protein was observed in the culture supernatant, a large amount of the full-length pelB-strep-P14K and pelB-α subunit (the pelB signal peptides were not cut off in either) were detected in the cell-free extract (Figure 5A). In addition, the enzyme activity in the cell-free extract was comparable to that of the wild-type NHase.

Figure 5
figure 5

SDS-PAGE analysis of recombinant pelB-strep-P14K. (A) SDS-PAGE analysis of the activators encoded by A(strep-P) and (pelB-A)B(pelB-strep-P). 1, mark; 2, cell extracts of the transformant containing pET-A(strep-P); 3, cell extracts of the transformant containing pET-(pelB-A)B(pelB-strep-P); 4, culture supernatant of the transformant containing pET-(pelB-A)B(pelB-strep-P). (B) SDS-PAGE analysis of stability of purified activator complexes [α-(strep-P14K)] and [α-(pelB-strep-P14K)]. 1, mark; 2, purified [α-(strep-P14K)]; 3, purified [α-(strep-P14K)] after storage for 1 day; 4, purified [α-(strep-P14K)] after storage for 2 days; 5, purified [α-(pelB-strep-P14K)]; 6, purified [α-(pelB-strep-P14K)] after storage for 2 days; 7, purified [α-(pelB-strep-P14K)] after storage for 6 days.

Full size image

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.

Conclusions

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.

Methods

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

The genomic DNA of P. putida NRRL-18668 was isolated for ABP cloning with the primers A-up and P-down (Table 2 and Figure 3). The PCR products were digested and ligated into pET-24a(+) to generate the plasmid pET-ABP. The recombinant plasmid pET-ABP was then transformed into E. coli BL21 (DE3) for heterologous expression. An overlap extension PCR protocol was used for construction of the plasmid pET-AB’P (’represents an insertion of RBS sequence) through two rounds of PCR. The first round of PCR was performed using the primer pairs A-up and B-down(rbs) and P-up(rbs) and P-down, respectively, the plasmid pET-ABP was used as the template. The second round of PCR was performed to produce the full-length AB’P, using the primers A-up and P-down and mixing equal molar amounts of the first-round products as template. The following plasmids were prepared similar to pET-AB’P. The plasmid pET-PAB was prepared with the primer pairs P-up and P-down(PAB) and A-up(PAB) and B-down(PAB). The plasmid pET-ABPo was constructed with the primer pairs A-up and B-down(rbs) and Po-up and Po-down. The plasmid pET-Po (Po, codon optimized P gene synthesized by Sangon Biotech Ltd.) was used as the template. The plasmid pET-AB(strep-P) was constructed with the primer pairs A-up and B-down(strep) and P-up(strep) and P-down as described previously [18]. The plasmid pET-AB(△N-P) was constructed with the primers A-up and B-down(rbs) and P-up(△N) and P-down. The plasmid pET-A(strep-P) was generated from pET-ABP with the primers A-up and A-down(strep) and P-up(strep) and P-down. The plasmid pET22b-(pelB-A)B(pelB-strep-P) was prepared with the primers of secA-up and secB-down and secP-up and P-down. The plasmid pET-ABP(K2A) mutant was constructed to produce full-length ABP(K2A) with the primers K2A-up and K2A-down using pET-AB’P as the template. The PCR products were digested with DpnI to degrade the template plasmid and then transformed into E. coli BL21 (DE3).

Table 2 Oligonucleotide primers used in this study
Full size table

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 [13]. 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.

References

  1. Noguchi T, Nojiri M, Takei K, Odaka M, Kamiya N: Protonation structures of Cys-sulfinic and Cys-sulfenic acids in the photosensitive nitrile hydratase revealed by Fourier transform infrared spectroscopy. Biochemistry. 2003, 42: 11642-11650. 10.1021/bi035260i.

    Article  CAS  Google Scholar 

  2. Kobayashi M, Shimizu S: Cobalt proteins. Eur J Biochem. 1999, 261: 1-9. 10.1046/j.1432-1327.1999.00186.x.

    Article  CAS  Google Scholar 

  3. Kobayashi M, Fujiwara Y, Goda M, Komeda H, Shimizu S: Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes. Proc Natl Acad Sci U S A. 1997, 94: 11986-11991. 10.1073/pnas.94.22.11986.

    Article  CAS  Google Scholar 

  4. Nagashima S, Nakasako M, Dohmae N, Tsujimura M, Takio K, Odaka M, Yohda M, Kamiya N, Endo I: Novel non-heme iron center of nitrile hydratase with a claw setting of oxygen atoms. Nat Struct Mol Biol. 1998, 5: 347-351. 10.1038/nsb0598-347.

    Article  CAS  Google Scholar 

  5. Murakami T, Nojiri M, Nakayama H, Dohmae N, Takio K, Odaka M, Endo I, Nagamune T, Yohda M: Post‒translational modification is essential for catalytic activity of nitrile hydratase. Protein Sci. 2000, 9: 1024-1030. 10.1110/ps.9.5.1024.

    Article  CAS  Google Scholar 

  6. Miyanaga A, Fushinobu S, Ito K, Shoun H, Wakagi T: Mutational and structural analysis of cobalt‒containing nitrile hydratase on substrate and metal binding. Eur J Biochem. 2003, 271: 429-438.

    Article  Google Scholar 

  7. Arakawa T, Kawano Y, Kataoka S, Katayama Y, Kamiya N, Yohda M, Odaka M: Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt (III) center. J Mol Biol. 2007, 366: 1497-1509. 10.1016/j.jmb.2006.12.011.

    Article  CAS  Google Scholar 

  8. Okamoto S, Eltis LD: The biological occurrence and trafficking of cobalt. Metallomics. 2011, 3: 963-970. 10.1039/c1mt00056j.

    Article  CAS  Google Scholar 

  9. Nojiri M, Yohda M, Odaka M, Matsushita Y, Tsujimura M, Yoshida T, Dohmae N, Takio K, Endo I: Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine. J Biochem. 1999, 125: 696-704. 10.1093/oxfordjournals.jbchem.a022339.

    Article  CAS  Google Scholar 

  10. Nishiyama M, Horinouchi S, Kobayashi M, Nagasawa T, Yamada H, Beppu T: Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23. J Bacteriol. 1991, 173: 2465-2472.

    CAS  Google Scholar 

  11. Hashimoto Y, Nishiyama M, Horinouchi S, Beppu T: Nitrile hydratase gene from Rhodococcus sp. N-774 requirement for its downstream region for efficient expression. Biosci Biotechnol Biochem. 1994, 58: 1859-1865. 10.1271/bbb.58.1859.

    Article  CAS  Google Scholar 

  12. Lu J, Zheng Y, Yamagishi H, Odaka M, Tsujimura M, Maeda M, Endo I: Motif CXCC in nitrile hydratase activator is critical for NHase biogenesis in vivo. FEBS Lett. 2003, 553: 391-396. 10.1016/S0014-5793(03)01070-6.

    Article  CAS  Google Scholar 

  13. Zhou Z, Hashimoto Y, Shiraki K, Kobayashi M: Discovery of posttranslational maturation by self-subunit swapping. Proc Natl Acad Sci U S A. 2008, 105: 14849-14854. 10.1073/pnas.0803428105.

    Article  CAS  Google Scholar 

  14. Zhou ZM, Hashimoto Y, Cui TW, Washizawa Y, Mino H, Kobayashi M: Unique Biogenesis of High-Molecular Mass Multimeric Metalloenzyme Nitrile Hydratase: Intermediates and a Proposed Mechanism for Self-Subunit Swapping Maturation. Biochemistry. 2010, 49: 9638-9648. 10.1021/bi100651v.

    Article  CAS  Google Scholar 

  15. Zhou ZM, Hashimoto Y, Kobayashi M: Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt. J Biol Chem. 2009, 284: 14930-14938. 10.1074/jbc.M808464200.

    Article  CAS  Google Scholar 

  16. Okamoto S, Van Petegem F, Patrauchan MA, Eltis LD: AnhE, a metallochaperone involved in the maturation of a cobalt-dependent nitrile hydratase. J Biol Chem. 2010, 285: 25126-25133. 10.1074/jbc.M110.109223.

    Article  CAS  Google Scholar 

  17. Wu S, Fallon RD, Payne MS: Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein. Appl Microbiol Biotechnol. 1997, 48: 704-708. 10.1007/s002530051119.

    Article  CAS  Google Scholar 

  18. Liu Y, Cui W, Xia Y, Cui Y, Kobayashi M, Zhou Z: Self-subunit swapping occurs in another gene type of cobalt nitrile hydratase. PLoS One. 2012, 7: e50829-10.1371/journal.pone.0050829.

    Article  CAS  Google Scholar 

  19. Varshavsky A: The N‒end rule pathway of protein degradation. Genes Cells. 1997, 2: 13-28. 10.1046/j.1365-2443.1997.1020301.x.

    Article  CAS  Google Scholar 

  20. Mogk A, Schmidt R, Bukau B: The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies. Trends Cell Biol. 2007, 17: 165-172. 10.1016/j.tcb.2007.02.001.

    Article  CAS  Google Scholar 

  21. Dougan D, Truscott K, Zeth K: The bacterial N‒end rule pathway: expect the unexpected. Mol Microbiol. 2010, 76: 545-558. 10.1111/j.1365-2958.2010.07120.x.

    Article  CAS  Google Scholar 

  22. Tobias JW, Shrader TE, Rocap G, Varshavsky A: The N-end rule in bacteria. Science. 1991, 254: 1374-1377. 10.1126/science.1962196.

    Article  CAS  Google Scholar 

  23. Dougan D, Micevski D, Truscott K: The N-end rule pathway: From recognition by N-recognins, to destruction by AAA + proteases. Biochim Biophys Acta. 1823, 2012: 83-91.

    Google Scholar 

  24. Salis HM, Mirsky EA, Voigt CA: Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol. 2009, 27: 946-950. 10.1038/nbt.1568.

    Article  CAS  Google Scholar 

  25. Icev A, Ruiz C, Ryder EF: Distance-enhanced association rules for gene expression. Gene. 2003, 10: 34-40.

    Google Scholar 

  26. Sahdev S, Khattar SK, Saini KS: Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem. 2008, 307: 249-264.

    Article  CAS  Google Scholar 

  27. Kwon WS, Da Silva NA, Kellis JT: Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli. Protein Eng. 1996, 9: 1197-1202. 10.1093/protein/9.12.1197.

    Article  CAS  Google Scholar 

  28. Choi J, Lee S: Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol. 2004, 64: 625-635. 10.1007/s00253-004-1559-9.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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).

Author information

Author notes

    Authors and Affiliations

    1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Peoples Republic of China

      Yi Liu, Wenjing Cui, Yuechun Yu, Youtian Cui, Yuanyuan Xia & Zhemin Zhou

    2. School of Environmental and Civil Engineering, Jiangnan University, Wuxi, 214122, Peoples Republic of China

      Yueqin Fang

    3. Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan

      Michihiko Kobayashi

    Authors
    1. Yi Liu
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Wenjing Cui
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Yueqin Fang
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Yuechun Yu
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Youtian Cui
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Yuanyuan Xia
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Michihiko Kobayashi
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Zhemin Zhou
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding authors

    Correspondence to Michihiko Kobayashi or Zhemin Zhou.

    Additional information

    Competing interests

    The authors declare that there are no competing interests.

    Authors’ contributions

    ZMZ, WJC and MK designed this study. YL, YQF, YCY, YTC and YYX performed the experimental work. ZMZ, WJC, YL and MK wrote the manuscript. ZMZ and MK collaborated in the coordination of the research and helped to draft the manuscript. All authors read and approved the submission of the manuscript.

    Yi Liu, Wenjing Cui contributed equally to this work.

    Authors’ original submitted files for images

    Rights and permissions

    Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Reprints and permissions

    About this article

    Cite this article

    Liu, Y., Cui, W., Fang, Y. et al. Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli. BMC Biotechnol 13, 48 (2013). https://doi.org/10.1186/1472-6750-13-48

    Download citation

    • Received:

    • Accepted:

    • Published:

    • DOI: https://doi.org/10.1186/1472-6750-13-48

    Keywords

    BMC Biotechnology

    ISSN: 1472-6750

    Contact us