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A Method for Efficient Expression of Pseudomonas aeruginosa Alginate Lyase in Pichia Pastoris a
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a
Michael M. Yue , Wendy W. Gong , Yu Qiao & Hongbiao Ding a
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Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China
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Institute Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China Accepted author version posted online: 08 Jan 2015.
Click for updates To cite this article: Michael M. Yue, Wendy W. Gong, Yu Qiao & Hongbiao Ding (2015): A Method for Efficient Expression of Pseudomonas aeruginosa Alginate Lyase in Pichia Pastoris, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.996233 To link to this article: http://dx.doi.org/10.1080/10826068.2014.996233
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A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris Michael M. Yue1, Wendy W. Gong2, Yu Qiao1, Hongbiao Ding1 1
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China, 2 Institute Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
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Address correspondence to Hongbiao Ding, Laboratory of feed biotechnology, Feed research institute, Chinese Academy of Agricultural Sciences, Zhongguancun South Rd. No.12, 100081 Beijing, China. E-mail: [email protected]
Abstract As an eco-friendly biocatalyst for alginate hydrolysis, bacteria-derived alginate lyase (AlgL) has been widely used in research and industries to produce oligosaccharides. However, the cost of AlgL enzyme production remains high due to the low expression and difficulty in purification from bacterial cells. In this study we report an effective method to overexpress the Pseudomonas aeruginosa AlgL (paAlgL) enzyme in Pichia pastoris. Fused with a secretory peptide, the recombinant paAlgL was expressed extracellularly and purified from the culture supernatant through a simple process. The purified recombinant enzyme is highly specific for alginate sodium with a maximal activity of 2,440 U/mg. The enzymatic activity remained stable below 45 °C and at pH between 4 and 10. The recombinant paAlgL was inhibited by Zn2+, Cu2+ and Fe2+ and promoted by Co2+ and Ca2+. Interestingly, we also found that the recombinant paAlgL significantly enhanced the antimicrobial activity of antibiotics, ampicillin and kanamycin, against Pseudomonas aeruginosa. Our results introduce a method for efficient AlgL
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production, the characterization and a new feature of the recombinant paAlgL as an enhancer of antibiotics against Pseudomonas aeruginosa.
KEYWORDS: Pseudomonas aeruginosa, alginate lyase, overexpression, Pichia pastoris, purification, antibiotic
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INTRODUCTION Alginate lyase, a biocatalyst that hydrolyzes alginate into mannuronic or guluronic oligosaccharides, has been found in various species, including algae, marine invertebrates, and microorganisms.[1] A number of crudely prepared alginate lyases from bacteria have been characterized and used in research and industries.[2, 3]
P. aeruginosa, a pathogenic bacterium, produces alginate and AlgL at different physiological stages. Genetic analysis has revealed that the P. aeruginosa algL (paalgL) gene is located in the alginate biosynthetic gene clusters,[4] indicating that AlgL plays a role in alginate biosynthesis.[5] As a major pathogenic factor in cystic fibrosis, alginate facilitates the attachment of the P. aeruginosa to patients’ tracheal epithelia tissue, resulting in resistance to antibiotics.[6] During the apoptosis phase, the overexpression of algL gene within a mucoid strain of P. aeruginosa led to a decrease in the length of alginate polymers and increased bacterial detachment from the surface.[7] Thus, the use of AlgL enzyme together with antibiotics may be feasible for therapy of infection caused by P. aeruginosa.[8]
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The paalgL gene was first cloned and expressed in E. coli.[9] Since then, other algL genes from different spices, including other Pseudomonas subtypes,[10,11] Azotobacter chroococcum,[12] Sphingomonas sp. A1,[13] Streptomyces sp.,[14] Vibrio sp.[15] and abalone,[16] had also been cloned and expressed in E. coli. In E. coli, however, the recombinant proteins tended to be insoluble[16] and require complicated purification
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procedures[12] which led to significant decrease in yield.
In this study, we demonstrate a strategy to efficiently produce the recombinant paAlgL. This is the first report about the overexpresstion and purification of paAlgL in Pichia pastoris. We also characterized the recombinant paAlgL and its role as an enhancer of antibiotics against Pseudomonas aeruginosa.
MATERIALS AND METHODS Strains P. aeruginosa strain was obtained from the China General Microbiological Culture Collection Center (CGMCC1.1785) and grown in nutrient broth (NB) medium (1% peptone, 0.3% beef extract, and 0.5% NaCl) at 37 °C. E. coli Top10 strain (lab stock) was grown in Luria-Bertani (LB) medium at 37 °C. The P. pastoris GS115 strain and the pPIC9K vector were purchased from Invitrogen. RDB, YPD, BMGY and BMMY media were used for P. pastoris GS115 growth as described in the manufacturer’s handbook (Pichia Expression Kit, Invitrogen).
Gene Cloning And Vector Construction
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The genomic DNA of P. aeruginosa was extracted by alkaline lysis method. The DNA sequence encoding the mature paAlgL region was amplified by PCR using Pfu DNA polymerase (Tiangen, China) and primers, paAlgL F 5’-GAG GAG AAT TCG CCG ACC TGG TAC CCC CGC C-3’ and paAlgL R 5’-GAG GAG AAT TCA CTT CCC CCT TCG CGG CTG AAC ACC-3’ (EcoR I site is underlined).
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To add a hexahistidine tag (6×his tag) to the C-terminus of paAlgL , two oligos containig restriction enzyme sites (SnaB I, EcoR I, Avr II and Not I) were synthesized: oligo 1: 5’-GTA GAA TTC CCT AGG CAT CAT CAT CAT CAT CAT GC-3’; oligo 2: 3’-CAT CTT AAG GGA TCC GTA GTA GTA GTA GTA GTA CGC CGG-5’. These two oligos were annealed gradually in a thermal cycler ramping from 72 °C to 20 °C. The annealed double-strand adapter was digested by SnaB I and Not I and inserted into the digested pPIC9K vector to generate the pPIC9K-6HIS vector.
The PCR product of mature paAlgL-coding sequence was digested by EcoR I and inserted into the digested pPIC9K-6HIS vector to generate the pPIC9K-algL-6HIS vector construct. The insert was verified by PCR using AOX5’ (5’-GAC TGG TTC CAA TTG ACA AGC-3’) and paAlgL R primers. The construct was then sequenced to confirm that the insert was in frame with the α-factor secretory peptide.
Protein Expression And Purification The linearized pPIC9K-algL-6HIS plasmid was transformed into P. pastoris GS115 by and selected on YPD plates containing geneticin (Invitrogen, USA) at different
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concentrations. The selected positive colonies were inoculated into 3 ml BMGY medium and grown at 30 °C. After 48 hours, the cells were transferred into 2 ml BMMY medium containing 0.5% methanol for induction. After another 48 hours, the culture supernatant was spotted into the wells on agarose plates containing 1% alginate sodium (Sigma, USA) and the plates were incubated at 37°C for 1 hour. According to the method described previously,[12] the plates were then stained by flooding with 10% (w/v) cetylpyridinium
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chloride, and clear zones of depolymerization on a white background were observed, indicating lyase activity. The colony showing the highest hydrolytic activity was selected for the induction in 100 ml BMMY medium (0.5% methanol was added every 24 hours) at 30 °C for 96 hours in the following experiments.
After induction, the cell pellets were removed from the cell culture supernatant. The protein in supernatant was precipitated by adding ammonium sulfate to a final concentration of 70% (w/v). The protein precipitate was dissolved in binding buffer (50 mM KH2PO4-K2HPO4, 500 mM NaCl, pH 8) and loaded onto a column packed with 1 ml of His•Bind Resin (Novagen, Germany). The column was extensively washed and then eluted by imidazole gradient in binding buffer.
Enzymatic Characterization The enzymatic reaction was performed by incubating 100 μl of enzyme solution with 100 μl of 10 mg/ml alginate sodium at 40 °C for 10 min and the enzymatic activity was measured by a photospectrometer at wavelength of 550 nm after addition of 100 μl
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of 1% 3, 5-dinitrosalicylic acid (DNS). One unit of the enzyme activity is defined as the amount of enzyme required to produce 1 μg reducing sugars per minute.
To determine the optimal pH, the purified recombinant paAlgL was diluted in different buffers (citric acid-K2HPO4, pH 2.2~5; KH2PO4-K2HPO4, pH5~8.5; KH2PO4-KOH, pH 8.5~12) and enzyme activity was then measured as described above.
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For the effect of pH on paAlgL stability, the purified recombinant paAlgL was diluted in the same buffers as mentioned above and incubated at 35 °C for 30 min before activity measurement. For the effect of temperature on the enzyme activity, the purified paAlgL (100 μg/ml) was incubated at different temperatures between 20 and 60 °C for 30 min and the residue paAlgL activity was determined at pH8.5 and 40 °C. The effect of metal ions on AlgL activity was determined in the presences of the ions (Zn2+, Mn2+, Cu2+, Mg2+, Fe2+, Co2+, Ca2+, Ka+ and Na+) at 1, 10 and 50 mM, respectively.
The substrate specificity of the recombinant paAlgL was determined after incubating the enzyme protein in 50 mM KH2PO4-K2HPO4 buffer containing 10 mg/ml of different substrates (alginate sodium, caboxymethylcellulose, xylan, pectin, soluble starch and locust beam gum) at pH 8.5 and 40 °C for 10 min. Km and Vmax were determined in 50 mM KH2PO4-K2HPO4 buffer containing 2~40 mg/ml alginate sodium after incubating with the purified paAlgL at pH8.5 and 40 °C for 5 min.
Antimicrobial Assay
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P. aeruginosa was grown at 37 °C in 20 ml NB medium until OD600 reached 0.6. 500 μl of cell culture was added into 100 ml pre-warmed (45 °C) NB medium containing 1.0% (w/v) agar and the medium was then poured into 100 mm-diameter Petri dishes with Oxford cups to form wells. Different amount of purified enzyme was mixed with 20 μg/ml ampicillin or 2 μg/ml kanamycin (in a final volume of 100 μl) and loaded into the wells, and the plates were incubated at 37 °C for 12 hours. The diameters of clear zone
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around the wells were measured.
RESULTS Expression Vector Construction The cloned paalgL gene was sequenced and found to be 99% identical with the sequences in GenBank (L14597 and U27829). The mature paAlgL coding sequence is a 1,023 bp fragment encoding 340 amino acids. In order to be led by the α-factor secretory peptide in the expression vector, the original AlgL signal peptide coding sequence was not included. The 6×his-tag coding fragment was correctly inserted into the pPIC9K plasmid and fused at the 3’ end of paalgL gene (Fig. 1).
Expression And Purification Of The Recombinant Proteins In order to screen for strains that may potentially harbor multi-copy number of paalgL gene,[17] the transformed P. pastoris colonies were selected on YPD plates containing geneticin (Fig. 2A). The colonies with high resistance to geneticin (> 3 mg/ml) were selected for further analysis (Fig. 2A). Positive yeast colonies were also tested on alginate-agarose plates (Fig. 2B). One colony showed the largest clear zone was selected
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and induced for purification in 100 ml culture. After transferring the yeast cells from BMGY medium to BMMY, the protein expression was induced by 0.5% methanol. Time course showed that highest enzymatic activity occurred on day 4 (96 hours post-induction) (Fig. 3B), although the secreted protein level still increased thereafter (Fig. 3A).
The protein in the culture supernatant was purified as summarized (Table 1). The
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purified recombinant AlgL yielded a single band with a molecular weight (Mr) of 39 kDa, as determined by SDS-PAGE (Fig. 4A). The specific activity of purified AlgL was 2,440 U/mg with a recovery rate of 40% after purification. The same Mr was observed after the treatment with Endo H, indicating that AlgL is not glycosylated (Fig. 4A).
Characterization Of The Purified Algl The purified paAlgL showed the highest activity at pH 8.5 (Fig. 4B) and 40 °C (Fig. 4D). The protein stability retained over the range of pH 4~11 (Fig. 4C). The enzyme was stable below 45 °C and inactivated after the incubation at 50 °C for 30 min (Fig. 4E). The tested metal ions had different effects on recombinant AlgL activity (Fig. 4F). The activity was increased by more than 20 % in the presence of 50 mM Mn2+, Mg2+, K+ or Na2+. Co2+ and Ca2+ increased the activity by 90 % and 50 %, respectively. The activity was significantly inhibited in the presence of 50 mM Cu2+ and Fe2+.
The purified AlgL exhibited an exclusively hydrolytic activity on alginate sodium, not on other polysaccharide substrates (data not shown). The recombinant paAlgL yielded a Km value of 4.8 mg/ml and a Vmax value of 1,143 U/min·mg for alginate sodium.
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Antimicrobial Activity Assay The effect of recombinant AlgL on antimicrobial activity of antibiotics against P. aeruginosa was determined by inhibition zone assay. The assay showed that the antimicrobial activity against P. aeruginosa was significant enhanced in the presence of the recombinant paAlgL enzyme (Fig. 5). When a final AlgL concentration of 40 μg/ml was used, the antimicrobial activities of ampicillin (Fig. 5A) and kanamycin (Fig. 5B)
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were increased by about 39% and 24% compared with the samples containing only antibiotics, respectively.
DISCUSSION The methylotrophic yeast, P. pastoris, has been developed as a high-level expression system because of its advantages for overproduction of heterologous proteins, and a number of active enzymes have been successfully expressed in this system.[18] Notably, the main expression product is secreted from P. pastoris, unlike in E. coli, from which the majority of protein tends to be expressed in inclusion bodies, and thus, leading to great loss of protein and low enzymatic activity. Our result demonstrates that P. pastoris system fits AlgL expression well and is superior to E. coli system. In previous studies, the full-length AlgL harboring its native signal peptide has been expressed in E. coli intracellularly.[9, 12, 13, 19] By using the P. pastoris system, we introduced a yeast recognizable secretory signal peptide, α-factor signal peptide, which facilitates the expressed protein to be secreted into the culture medium. The multiple-step purification of AlgL from E. coli cells yielded only 10.9% of the original activity,[13] whereas in our study we achieved a recovery rate at 40% after a one-step purification. In addition,
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compared with the AlgL expression using the original pPIC9K vector in our previous work (data not shown), the AlgL activity was not affected by the addition of a 6×his-tag at C-terminus. By this optimization, the fused AlgL could be purified with Ni2+ ion affinity resin in a single step, which minimizes the protein loss during the purification process.
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The characterization results from the tests using purified AlgL protein appear to be more reliable than a crude preparation. The specific activity of the purified AlgL is 2,440 U/mg, which is higher than an AlgL from a marine bacterium Vibrio sp. 510-64 measured by the same DNS method.[20] The purified AlgL has an optimal pH of 8.5 and an optimal temperature of 40 °C, which are similar to the enzymes from the native strains, P. aeruginosa[1,19] and A. chroococcum[12], and the recombinant AlgL expressed in E.coli[12]. In addition, the recombinant AlgL from P. pastoris was found to be stable over a broad range of pH, suggesting this recombinant enzyme may be suitable for different applications under various conditions.
We found that the activity of the recombinant AlgL was enhanced by Co2+, Ca2+, Mn2+, Mg2+, K+ and Na+. This is consistent with the AlgL from A. chroococcum[12] and K. aerogenes[1], whose activities need Mg2+, K+ and Na2+, and that from Bacillus sp. ATB-1015[21] which needs Co2+, Ca2+ and Mn2+. It was shown that the enzyme promoted by Na+ may be partly caused by removal of bound water from sodium alginate molecules or by the effects of charge in forming the alginate-enzyme complex.[1] In the meanwhile, the presence of divalent cations (such as Ca2+ or Mg2+) is required for the optimal
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enzymatic activity of AlgL.[1] In our study, we also found that the enzyme activity was suppressed by Cu2+ and Fe2+, possibly by blocking the catalytic center of the enzyme or by their binding to the enzyme proteins when these cations presented at a high concentration, 50 mM, therefore, inducing precipitation of the enzyme and leading to the loss of activity.
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The present data suggest that the antimicrobial activity of ampicillin and kanamycin agaist P. aeruginosa was increased when AlgL was used. These results are consistent with the observation that AlgL could enhance the antimicrobial effects of tobramycin and gentamicin.[22] Due to the degradation of the extracellular alginate biofilm by AlgL, the diffusion of antibiotics to the target bacteria can be enhanced. The substrate specificity of the recombinant paAlgL to alginate suggests that the recombinant enzyme can be an effective enhancer in the therapy of infectious diseases caused by P. aeruginosa.
CONCLUSIONS Our study provides an effective method for secretary expression and purification of alginate lyase in yeast. This strategy can be a powerful tool for AlgL preparation and facilitate the production of bioactive alginate oligosaccharides and therapeutic development for the diseases caused by Pseudomonas aeruginosa.
FUNDING This research was supported by the National High Technology Research and Development Program of China (Grant No. 2013AA102801).
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Microbiol 2009, 48(1), 38-43. 3. Wang, Y.H.; Yu, G.L.; Wang, X.M.; Lv, Z.H.; Zhao, X.; Wu, Z.H.; Ji, W.S. Purification and characterization of alginate lyase from marine Vibrio sp. YWA. Acta Biochim Biophys Sin (Shanghai) 2006, 38(9), 633-638. 4. Chitnis, C. E.; Ohman, D. E. Genetic analysis of the alginate biosynthetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic structure. Mol Microbiol 1993, 8(3), 583-593. 5. Albrecht, M.T.; Schiller, N.L. Alginate lyase (AlgL) activity is required for alginate biosynthesis in Pseudomonas aeruginosa. J Bacteriol 2005, 187(11), 3869-3872. 6. Boyd, A.; Chakrabarty, A.M. Role of alginate lyase in cell detachment of Pseudomonas aeruginosa. Appl Environ Microbiol 1994, 60(7), 2355-2359. 7. Jain, S.; Ohman, D.E. Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa. Infect Immun 2005, 73(10), 6429-6436. 8. Alkawash, M.A.; Soothill, J.S.; Schiller, N.L. Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS 2006, 114(2), 131-138.
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9. Boyd, A.; Ghosh, M.; May. T.B.; Shinabarger, D.; Keogh, R.; Chakrabarty, A.M. Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product. Gene 1993, 131(1), 1-8. 10. Preston, L.A.; Bender, C.L.; Schiller, N.L. Analysis and expression of algL, which encodes alginate lyase in Pseudomonas syringae Pv. syringae. DNA Seq 2001, 12(5-6), 455-461.
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11. Sawabe, T.; Takahashi, H.; Ezura, Y.; Gacesa, P. Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydr Res 2001, 335(1), 11-21. 12. Pecina, A.; Pascual, A.; Paneque, A. Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme. J Bacteriol 1999, 181(5), 1409-1414. 13. Miyake, O.; Hashimoto, W.; Murata, K. An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV). Protein Expr Purif 2003, 29(1), 33-41. 14. Kim, D.E.; Lee, E.Y.; Kim, H.S. Cloning and characterization of alginate lyase from a marine bacterium Streptomyces sp. ALG-5. Mar Biotechnol (NY) 2009, 11(1), 10-16. 15. Kawamoto, H.; Horibe, A.; Miki, Y.; Kimura, T.; Tanaka, K.; Nakagawa, T.; Kawamukai, M.; Matsuda, H. Cloning and sequencing analysis of alginate lyase genes from the marine bacterium Vibrio sp. O2. Mar Biotechnol (NY) 2006, 8(5), 481-490. 16. Shimizu, E.; Ojima, T.; Nishita, K. cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai. Carbohydr Res 2003, 338(24), 2841-2852.
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17. Scorer, C.A.; Clare, J.J.; McCombie, W.R.; Romanos, M.A.; Sreekrishna, K. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology (N Y) 1994, 12(2), 181-184. 18. Macauley-Patrick, S.; Fazenda, M.L.; McNeil, B.; Harvey, L.M. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005, 22(4), 249-270.
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19. Schiller, N.L.; Monday, S.R.; Boyd, C.M.; Keen, N.T.; Ohman, D.E. Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J Bacteriol 1993, 175(15), 4780-4789. 20. Hu, X.; Jiang, X.; Hwang, H.M. Purification and characterization of an alginate lyase from marine Bacterium Vibrio sp. mutant strain 510-64. Curr Microbiol 2006, 53(2), 135-140. 21. Nakagawa, A.; Ozaki, T.; Chubachi, K.; Hosoyama, T.; Okubo, T.; Iyobe, S.; Suzuki, T. An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase. J Appl Microbiol 1998, 84(3), 328-335. 22. Hatch, R.A.; Schiller, N.L. Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa. Antimicrob Agents Chemother 1998, 42(4), 974-977.
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Table 1. Summary of purification procedure for recombinant AlgL Purification steps
Volume Total
Total
Specific
Purified
(ml)
protein
activity
activity
fold
(mg)
(U)
(U/mg)
Culture supernatant
40
6.52
5,499
843
1
(NH4)2SO4
35
3.9
4,508
1,156
1.37
20
0.91
2,220
2,440
2.89
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precipitation Fusion protein
15
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Figure 1. Schematic of the expression vector construction.
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Figure 2. Selection of paAlgL expressing yeast colonies. (A) Selection of paalgL integrants on YPD plates containing Geneticin at different concentrations. (B) Selection of AlgL expressing colonies on alginate-agarose plates by clear zone assay. The arrows
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indicate the positive colonies.
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Figure 3. Expression of paAlgL in P. pastoris. (A) SDS-PAGE analysis of paAlgL expressed in P. pastoris in 100 ml medium. Lanes 1~9, culture supernatants after 24, 36, 48, 60, 72, 96, 120, 144, and 168 hours of induction. The arrow indicates the expressed recombinant paAlgL. All the protein samples were loaded onto a 12.5% poly acrylamide gel under denaturing conditions. The gel was stained with Coomassie Brilliant Blue R-250. (B) Expression time course of recombinant paAlgL. Enzymatic activity was
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measured using the culture supernatants.
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Figure 4. Purification and characterization of the recombinant paAlgL protein. (A) SDS-PAGE analysis of purified and deglycosylated AlgL. Lane 1, precipitated protein sample from the culture supernatant; lane 2, purified AlgL; lane 3, AlgL deglycosylated by Endo H. (B) The effect of pH on paAlgL activity. The arrow indicates the optimal pH. (C) The effect of pH on paAlgL stability. (D) The effect of temperature on paAlgL activity. The arrow indicates the optimal temperature. (E) The effect of temperature on
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paAlgL stability. (F) The effect of metal ions on paAlgL activity. The relative enzymatic activity was calculated and shown in percentages. Quantitative data are presented as the mean ± SD and compared statistically by Student's t-test.
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Figure 5. Enhancement of antimicrobial activity of antibiotics against P. aeruginosa. (A) Antimicrobial assay of ampicillin (Amp). Well 1, 100 μl Amp (20 μg/ml); well 2, 100 μl mixture of Amp (20 μg/ml) and AlgL (40 μg/ml); well 3, 100 μl AlgL (40 μg/ml). (B) Antimicrobial assay of kanamycin (Kan). well 1, 100 μl Kan (2 μg/ml); well 2, 100 μl
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mixture of Kan (2 μg/ml) and AlgL (40 μg/ml); well 3, 100 μl AlgL (40 μg/ml).
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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
A Method for Efficient Expression of Pseudomonas aeruginosa Alginate Lyase in Pichia Pastoris a
b
a
Michael M. Yue , Wendy W. Gong , Yu Qiao & Hongbiao Ding a
a
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China
b
Institute Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China Accepted author version posted online: 08 Jan 2015.
Click for updates To cite this article: Michael M. Yue, Wendy W. Gong, Yu Qiao & Hongbiao Ding (2015): A Method for Efficient Expression of Pseudomonas aeruginosa Alginate Lyase in Pichia Pastoris, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.996233 To link to this article: http://dx.doi.org/10.1080/10826068.2014.996233
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A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris Michael M. Yue1, Wendy W. Gong2, Yu Qiao1, Hongbiao Ding1 1
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China, 2 Institute Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
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Address correspondence to Hongbiao Ding, Laboratory of feed biotechnology, Feed research institute, Chinese Academy of Agricultural Sciences, Zhongguancun South Rd. No.12, 100081 Beijing, China. E-mail: [email protected]
Abstract As an eco-friendly biocatalyst for alginate hydrolysis, bacteria-derived alginate lyase (AlgL) has been widely used in research and industries to produce oligosaccharides. However, the cost of AlgL enzyme production remains high due to the low expression and difficulty in purification from bacterial cells. In this study we report an effective method to overexpress the Pseudomonas aeruginosa AlgL (paAlgL) enzyme in Pichia pastoris. Fused with a secretory peptide, the recombinant paAlgL was expressed extracellularly and purified from the culture supernatant through a simple process. The purified recombinant enzyme is highly specific for alginate sodium with a maximal activity of 2,440 U/mg. The enzymatic activity remained stable below 45 °C and at pH between 4 and 10. The recombinant paAlgL was inhibited by Zn2+, Cu2+ and Fe2+ and promoted by Co2+ and Ca2+. Interestingly, we also found that the recombinant paAlgL significantly enhanced the antimicrobial activity of antibiotics, ampicillin and kanamycin, against Pseudomonas aeruginosa. Our results introduce a method for efficient AlgL
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production, the characterization and a new feature of the recombinant paAlgL as an enhancer of antibiotics against Pseudomonas aeruginosa.
KEYWORDS: Pseudomonas aeruginosa, alginate lyase, overexpression, Pichia pastoris, purification, antibiotic
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INTRODUCTION Alginate lyase, a biocatalyst that hydrolyzes alginate into mannuronic or guluronic oligosaccharides, has been found in various species, including algae, marine invertebrates, and microorganisms.[1] A number of crudely prepared alginate lyases from bacteria have been characterized and used in research and industries.[2, 3]
P. aeruginosa, a pathogenic bacterium, produces alginate and AlgL at different physiological stages. Genetic analysis has revealed that the P. aeruginosa algL (paalgL) gene is located in the alginate biosynthetic gene clusters,[4] indicating that AlgL plays a role in alginate biosynthesis.[5] As a major pathogenic factor in cystic fibrosis, alginate facilitates the attachment of the P. aeruginosa to patients’ tracheal epithelia tissue, resulting in resistance to antibiotics.[6] During the apoptosis phase, the overexpression of algL gene within a mucoid strain of P. aeruginosa led to a decrease in the length of alginate polymers and increased bacterial detachment from the surface.[7] Thus, the use of AlgL enzyme together with antibiotics may be feasible for therapy of infection caused by P. aeruginosa.[8]
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The paalgL gene was first cloned and expressed in E. coli.[9] Since then, other algL genes from different spices, including other Pseudomonas subtypes,[10,11] Azotobacter chroococcum,[12] Sphingomonas sp. A1,[13] Streptomyces sp.,[14] Vibrio sp.[15] and abalone,[16] had also been cloned and expressed in E. coli. In E. coli, however, the recombinant proteins tended to be insoluble[16] and require complicated purification
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procedures[12] which led to significant decrease in yield.
In this study, we demonstrate a strategy to efficiently produce the recombinant paAlgL. This is the first report about the overexpresstion and purification of paAlgL in Pichia pastoris. We also characterized the recombinant paAlgL and its role as an enhancer of antibiotics against Pseudomonas aeruginosa.
MATERIALS AND METHODS Strains P. aeruginosa strain was obtained from the China General Microbiological Culture Collection Center (CGMCC1.1785) and grown in nutrient broth (NB) medium (1% peptone, 0.3% beef extract, and 0.5% NaCl) at 37 °C. E. coli Top10 strain (lab stock) was grown in Luria-Bertani (LB) medium at 37 °C. The P. pastoris GS115 strain and the pPIC9K vector were purchased from Invitrogen. RDB, YPD, BMGY and BMMY media were used for P. pastoris GS115 growth as described in the manufacturer’s handbook (Pichia Expression Kit, Invitrogen).
Gene Cloning And Vector Construction
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The genomic DNA of P. aeruginosa was extracted by alkaline lysis method. The DNA sequence encoding the mature paAlgL region was amplified by PCR using Pfu DNA polymerase (Tiangen, China) and primers, paAlgL F 5’-GAG GAG AAT TCG CCG ACC TGG TAC CCC CGC C-3’ and paAlgL R 5’-GAG GAG AAT TCA CTT CCC CCT TCG CGG CTG AAC ACC-3’ (EcoR I site is underlined).
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To add a hexahistidine tag (6×his tag) to the C-terminus of paAlgL , two oligos containig restriction enzyme sites (SnaB I, EcoR I, Avr II and Not I) were synthesized: oligo 1: 5’-GTA GAA TTC CCT AGG CAT CAT CAT CAT CAT CAT GC-3’; oligo 2: 3’-CAT CTT AAG GGA TCC GTA GTA GTA GTA GTA GTA CGC CGG-5’. These two oligos were annealed gradually in a thermal cycler ramping from 72 °C to 20 °C. The annealed double-strand adapter was digested by SnaB I and Not I and inserted into the digested pPIC9K vector to generate the pPIC9K-6HIS vector.
The PCR product of mature paAlgL-coding sequence was digested by EcoR I and inserted into the digested pPIC9K-6HIS vector to generate the pPIC9K-algL-6HIS vector construct. The insert was verified by PCR using AOX5’ (5’-GAC TGG TTC CAA TTG ACA AGC-3’) and paAlgL R primers. The construct was then sequenced to confirm that the insert was in frame with the α-factor secretory peptide.
Protein Expression And Purification The linearized pPIC9K-algL-6HIS plasmid was transformed into P. pastoris GS115 by and selected on YPD plates containing geneticin (Invitrogen, USA) at different
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concentrations. The selected positive colonies were inoculated into 3 ml BMGY medium and grown at 30 °C. After 48 hours, the cells were transferred into 2 ml BMMY medium containing 0.5% methanol for induction. After another 48 hours, the culture supernatant was spotted into the wells on agarose plates containing 1% alginate sodium (Sigma, USA) and the plates were incubated at 37°C for 1 hour. According to the method described previously,[12] the plates were then stained by flooding with 10% (w/v) cetylpyridinium
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chloride, and clear zones of depolymerization on a white background were observed, indicating lyase activity. The colony showing the highest hydrolytic activity was selected for the induction in 100 ml BMMY medium (0.5% methanol was added every 24 hours) at 30 °C for 96 hours in the following experiments.
After induction, the cell pellets were removed from the cell culture supernatant. The protein in supernatant was precipitated by adding ammonium sulfate to a final concentration of 70% (w/v). The protein precipitate was dissolved in binding buffer (50 mM KH2PO4-K2HPO4, 500 mM NaCl, pH 8) and loaded onto a column packed with 1 ml of His•Bind Resin (Novagen, Germany). The column was extensively washed and then eluted by imidazole gradient in binding buffer.
Enzymatic Characterization The enzymatic reaction was performed by incubating 100 μl of enzyme solution with 100 μl of 10 mg/ml alginate sodium at 40 °C for 10 min and the enzymatic activity was measured by a photospectrometer at wavelength of 550 nm after addition of 100 μl
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of 1% 3, 5-dinitrosalicylic acid (DNS). One unit of the enzyme activity is defined as the amount of enzyme required to produce 1 μg reducing sugars per minute.
To determine the optimal pH, the purified recombinant paAlgL was diluted in different buffers (citric acid-K2HPO4, pH 2.2~5; KH2PO4-K2HPO4, pH5~8.5; KH2PO4-KOH, pH 8.5~12) and enzyme activity was then measured as described above.
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For the effect of pH on paAlgL stability, the purified recombinant paAlgL was diluted in the same buffers as mentioned above and incubated at 35 °C for 30 min before activity measurement. For the effect of temperature on the enzyme activity, the purified paAlgL (100 μg/ml) was incubated at different temperatures between 20 and 60 °C for 30 min and the residue paAlgL activity was determined at pH8.5 and 40 °C. The effect of metal ions on AlgL activity was determined in the presences of the ions (Zn2+, Mn2+, Cu2+, Mg2+, Fe2+, Co2+, Ca2+, Ka+ and Na+) at 1, 10 and 50 mM, respectively.
The substrate specificity of the recombinant paAlgL was determined after incubating the enzyme protein in 50 mM KH2PO4-K2HPO4 buffer containing 10 mg/ml of different substrates (alginate sodium, caboxymethylcellulose, xylan, pectin, soluble starch and locust beam gum) at pH 8.5 and 40 °C for 10 min. Km and Vmax were determined in 50 mM KH2PO4-K2HPO4 buffer containing 2~40 mg/ml alginate sodium after incubating with the purified paAlgL at pH8.5 and 40 °C for 5 min.
Antimicrobial Assay
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P. aeruginosa was grown at 37 °C in 20 ml NB medium until OD600 reached 0.6. 500 μl of cell culture was added into 100 ml pre-warmed (45 °C) NB medium containing 1.0% (w/v) agar and the medium was then poured into 100 mm-diameter Petri dishes with Oxford cups to form wells. Different amount of purified enzyme was mixed with 20 μg/ml ampicillin or 2 μg/ml kanamycin (in a final volume of 100 μl) and loaded into the wells, and the plates were incubated at 37 °C for 12 hours. The diameters of clear zone
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around the wells were measured.
RESULTS Expression Vector Construction The cloned paalgL gene was sequenced and found to be 99% identical with the sequences in GenBank (L14597 and U27829). The mature paAlgL coding sequence is a 1,023 bp fragment encoding 340 amino acids. In order to be led by the α-factor secretory peptide in the expression vector, the original AlgL signal peptide coding sequence was not included. The 6×his-tag coding fragment was correctly inserted into the pPIC9K plasmid and fused at the 3’ end of paalgL gene (Fig. 1).
Expression And Purification Of The Recombinant Proteins In order to screen for strains that may potentially harbor multi-copy number of paalgL gene,[17] the transformed P. pastoris colonies were selected on YPD plates containing geneticin (Fig. 2A). The colonies with high resistance to geneticin (> 3 mg/ml) were selected for further analysis (Fig. 2A). Positive yeast colonies were also tested on alginate-agarose plates (Fig. 2B). One colony showed the largest clear zone was selected
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and induced for purification in 100 ml culture. After transferring the yeast cells from BMGY medium to BMMY, the protein expression was induced by 0.5% methanol. Time course showed that highest enzymatic activity occurred on day 4 (96 hours post-induction) (Fig. 3B), although the secreted protein level still increased thereafter (Fig. 3A).
The protein in the culture supernatant was purified as summarized (Table 1). The
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purified recombinant AlgL yielded a single band with a molecular weight (Mr) of 39 kDa, as determined by SDS-PAGE (Fig. 4A). The specific activity of purified AlgL was 2,440 U/mg with a recovery rate of 40% after purification. The same Mr was observed after the treatment with Endo H, indicating that AlgL is not glycosylated (Fig. 4A).
Characterization Of The Purified Algl The purified paAlgL showed the highest activity at pH 8.5 (Fig. 4B) and 40 °C (Fig. 4D). The protein stability retained over the range of pH 4~11 (Fig. 4C). The enzyme was stable below 45 °C and inactivated after the incubation at 50 °C for 30 min (Fig. 4E). The tested metal ions had different effects on recombinant AlgL activity (Fig. 4F). The activity was increased by more than 20 % in the presence of 50 mM Mn2+, Mg2+, K+ or Na2+. Co2+ and Ca2+ increased the activity by 90 % and 50 %, respectively. The activity was significantly inhibited in the presence of 50 mM Cu2+ and Fe2+.
The purified AlgL exhibited an exclusively hydrolytic activity on alginate sodium, not on other polysaccharide substrates (data not shown). The recombinant paAlgL yielded a Km value of 4.8 mg/ml and a Vmax value of 1,143 U/min·mg for alginate sodium.
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Antimicrobial Activity Assay The effect of recombinant AlgL on antimicrobial activity of antibiotics against P. aeruginosa was determined by inhibition zone assay. The assay showed that the antimicrobial activity against P. aeruginosa was significant enhanced in the presence of the recombinant paAlgL enzyme (Fig. 5). When a final AlgL concentration of 40 μg/ml was used, the antimicrobial activities of ampicillin (Fig. 5A) and kanamycin (Fig. 5B)
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were increased by about 39% and 24% compared with the samples containing only antibiotics, respectively.
DISCUSSION The methylotrophic yeast, P. pastoris, has been developed as a high-level expression system because of its advantages for overproduction of heterologous proteins, and a number of active enzymes have been successfully expressed in this system.[18] Notably, the main expression product is secreted from P. pastoris, unlike in E. coli, from which the majority of protein tends to be expressed in inclusion bodies, and thus, leading to great loss of protein and low enzymatic activity. Our result demonstrates that P. pastoris system fits AlgL expression well and is superior to E. coli system. In previous studies, the full-length AlgL harboring its native signal peptide has been expressed in E. coli intracellularly.[9, 12, 13, 19] By using the P. pastoris system, we introduced a yeast recognizable secretory signal peptide, α-factor signal peptide, which facilitates the expressed protein to be secreted into the culture medium. The multiple-step purification of AlgL from E. coli cells yielded only 10.9% of the original activity,[13] whereas in our study we achieved a recovery rate at 40% after a one-step purification. In addition,
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compared with the AlgL expression using the original pPIC9K vector in our previous work (data not shown), the AlgL activity was not affected by the addition of a 6×his-tag at C-terminus. By this optimization, the fused AlgL could be purified with Ni2+ ion affinity resin in a single step, which minimizes the protein loss during the purification process.
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The characterization results from the tests using purified AlgL protein appear to be more reliable than a crude preparation. The specific activity of the purified AlgL is 2,440 U/mg, which is higher than an AlgL from a marine bacterium Vibrio sp. 510-64 measured by the same DNS method.[20] The purified AlgL has an optimal pH of 8.5 and an optimal temperature of 40 °C, which are similar to the enzymes from the native strains, P. aeruginosa[1,19] and A. chroococcum[12], and the recombinant AlgL expressed in E.coli[12]. In addition, the recombinant AlgL from P. pastoris was found to be stable over a broad range of pH, suggesting this recombinant enzyme may be suitable for different applications under various conditions.
We found that the activity of the recombinant AlgL was enhanced by Co2+, Ca2+, Mn2+, Mg2+, K+ and Na+. This is consistent with the AlgL from A. chroococcum[12] and K. aerogenes[1], whose activities need Mg2+, K+ and Na2+, and that from Bacillus sp. ATB-1015[21] which needs Co2+, Ca2+ and Mn2+. It was shown that the enzyme promoted by Na+ may be partly caused by removal of bound water from sodium alginate molecules or by the effects of charge in forming the alginate-enzyme complex.[1] In the meanwhile, the presence of divalent cations (such as Ca2+ or Mg2+) is required for the optimal
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enzymatic activity of AlgL.[1] In our study, we also found that the enzyme activity was suppressed by Cu2+ and Fe2+, possibly by blocking the catalytic center of the enzyme or by their binding to the enzyme proteins when these cations presented at a high concentration, 50 mM, therefore, inducing precipitation of the enzyme and leading to the loss of activity.
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The present data suggest that the antimicrobial activity of ampicillin and kanamycin agaist P. aeruginosa was increased when AlgL was used. These results are consistent with the observation that AlgL could enhance the antimicrobial effects of tobramycin and gentamicin.[22] Due to the degradation of the extracellular alginate biofilm by AlgL, the diffusion of antibiotics to the target bacteria can be enhanced. The substrate specificity of the recombinant paAlgL to alginate suggests that the recombinant enzyme can be an effective enhancer in the therapy of infectious diseases caused by P. aeruginosa.
CONCLUSIONS Our study provides an effective method for secretary expression and purification of alginate lyase in yeast. This strategy can be a powerful tool for AlgL preparation and facilitate the production of bioactive alginate oligosaccharides and therapeutic development for the diseases caused by Pseudomonas aeruginosa.
FUNDING This research was supported by the National High Technology Research and Development Program of China (Grant No. 2013AA102801).
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REFERENCES 1. Wong, T.Y.; Preston, L.A.; Schiller, N.L. ALGINATE LYASE: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol 2000, 54, 289-340. 2. Tang, J.C.; Taniguchi, H.; Chu, H.; Zhou, Q.; Nagata, S. Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes. Lett Appl
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Microbiol 2009, 48(1), 38-43. 3. Wang, Y.H.; Yu, G.L.; Wang, X.M.; Lv, Z.H.; Zhao, X.; Wu, Z.H.; Ji, W.S. Purification and characterization of alginate lyase from marine Vibrio sp. YWA. Acta Biochim Biophys Sin (Shanghai) 2006, 38(9), 633-638. 4. Chitnis, C. E.; Ohman, D. E. Genetic analysis of the alginate biosynthetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic structure. Mol Microbiol 1993, 8(3), 583-593. 5. Albrecht, M.T.; Schiller, N.L. Alginate lyase (AlgL) activity is required for alginate biosynthesis in Pseudomonas aeruginosa. J Bacteriol 2005, 187(11), 3869-3872. 6. Boyd, A.; Chakrabarty, A.M. Role of alginate lyase in cell detachment of Pseudomonas aeruginosa. Appl Environ Microbiol 1994, 60(7), 2355-2359. 7. Jain, S.; Ohman, D.E. Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa. Infect Immun 2005, 73(10), 6429-6436. 8. Alkawash, M.A.; Soothill, J.S.; Schiller, N.L. Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS 2006, 114(2), 131-138.
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9. Boyd, A.; Ghosh, M.; May. T.B.; Shinabarger, D.; Keogh, R.; Chakrabarty, A.M. Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product. Gene 1993, 131(1), 1-8. 10. Preston, L.A.; Bender, C.L.; Schiller, N.L. Analysis and expression of algL, which encodes alginate lyase in Pseudomonas syringae Pv. syringae. DNA Seq 2001, 12(5-6), 455-461.
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11. Sawabe, T.; Takahashi, H.; Ezura, Y.; Gacesa, P. Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydr Res 2001, 335(1), 11-21. 12. Pecina, A.; Pascual, A.; Paneque, A. Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme. J Bacteriol 1999, 181(5), 1409-1414. 13. Miyake, O.; Hashimoto, W.; Murata, K. An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV). Protein Expr Purif 2003, 29(1), 33-41. 14. Kim, D.E.; Lee, E.Y.; Kim, H.S. Cloning and characterization of alginate lyase from a marine bacterium Streptomyces sp. ALG-5. Mar Biotechnol (NY) 2009, 11(1), 10-16. 15. Kawamoto, H.; Horibe, A.; Miki, Y.; Kimura, T.; Tanaka, K.; Nakagawa, T.; Kawamukai, M.; Matsuda, H. Cloning and sequencing analysis of alginate lyase genes from the marine bacterium Vibrio sp. O2. Mar Biotechnol (NY) 2006, 8(5), 481-490. 16. Shimizu, E.; Ojima, T.; Nishita, K. cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai. Carbohydr Res 2003, 338(24), 2841-2852.
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17. Scorer, C.A.; Clare, J.J.; McCombie, W.R.; Romanos, M.A.; Sreekrishna, K. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology (N Y) 1994, 12(2), 181-184. 18. Macauley-Patrick, S.; Fazenda, M.L.; McNeil, B.; Harvey, L.M. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005, 22(4), 249-270.
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19. Schiller, N.L.; Monday, S.R.; Boyd, C.M.; Keen, N.T.; Ohman, D.E. Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J Bacteriol 1993, 175(15), 4780-4789. 20. Hu, X.; Jiang, X.; Hwang, H.M. Purification and characterization of an alginate lyase from marine Bacterium Vibrio sp. mutant strain 510-64. Curr Microbiol 2006, 53(2), 135-140. 21. Nakagawa, A.; Ozaki, T.; Chubachi, K.; Hosoyama, T.; Okubo, T.; Iyobe, S.; Suzuki, T. An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase. J Appl Microbiol 1998, 84(3), 328-335. 22. Hatch, R.A.; Schiller, N.L. Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa. Antimicrob Agents Chemother 1998, 42(4), 974-977.
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Table 1. Summary of purification procedure for recombinant AlgL Purification steps
Volume Total
Total
Specific
Purified
(ml)
protein
activity
activity
fold
(mg)
(U)
(U/mg)
Culture supernatant
40
6.52
5,499
843
1
(NH4)2SO4
35
3.9
4,508
1,156
1.37
20
0.91
2,220
2,440
2.89
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precipitation Fusion protein
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Figure 1. Schematic of the expression vector construction.
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Figure 2. Selection of paAlgL expressing yeast colonies. (A) Selection of paalgL integrants on YPD plates containing Geneticin at different concentrations. (B) Selection of AlgL expressing colonies on alginate-agarose plates by clear zone assay. The arrows
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indicate the positive colonies.
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Figure 3. Expression of paAlgL in P. pastoris. (A) SDS-PAGE analysis of paAlgL expressed in P. pastoris in 100 ml medium. Lanes 1~9, culture supernatants after 24, 36, 48, 60, 72, 96, 120, 144, and 168 hours of induction. The arrow indicates the expressed recombinant paAlgL. All the protein samples were loaded onto a 12.5% poly acrylamide gel under denaturing conditions. The gel was stained with Coomassie Brilliant Blue R-250. (B) Expression time course of recombinant paAlgL. Enzymatic activity was
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measured using the culture supernatants.
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Figure 4. Purification and characterization of the recombinant paAlgL protein. (A) SDS-PAGE analysis of purified and deglycosylated AlgL. Lane 1, precipitated protein sample from the culture supernatant; lane 2, purified AlgL; lane 3, AlgL deglycosylated by Endo H. (B) The effect of pH on paAlgL activity. The arrow indicates the optimal pH. (C) The effect of pH on paAlgL stability. (D) The effect of temperature on paAlgL activity. The arrow indicates the optimal temperature. (E) The effect of temperature on
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paAlgL stability. (F) The effect of metal ions on paAlgL activity. The relative enzymatic activity was calculated and shown in percentages. Quantitative data are presented as the mean ± SD and compared statistically by Student's t-test.
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Figure 5. Enhancement of antimicrobial activity of antibiotics against P. aeruginosa. (A) Antimicrobial assay of ampicillin (Amp). Well 1, 100 μl Amp (20 μg/ml); well 2, 100 μl mixture of Amp (20 μg/ml) and AlgL (40 μg/ml); well 3, 100 μl AlgL (40 μg/ml). (B) Antimicrobial assay of kanamycin (Kan). well 1, 100 μl Kan (2 μg/ml); well 2, 100 μl
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mixture of Kan (2 μg/ml) and AlgL (40 μg/ml); well 3, 100 μl AlgL (40 μg/ml).
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