Hydrophobilization of lysozyme by genetic combination with polyproline at the carboxyl terminal

Chemistry Letters

Volumn , Issue 1, 1997, Pages 65-66

  Ito, Yoshihiro ^a   Hamamatsu, Norio ^a   Kwon, Oh Hyeong ^a   Ueda, Mitsuyoshi ^a   Tanaka, Atsuo ^a   Imanishi, Yukio ^a  

^a KYOTO UNIVERSITY   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 0031477987     PISSN: 03667022     EISSN: None     Source Type: Journal    
DOI: 10.1246/cl.1997.65     Document Type: Article

References (17)

1. a) A. M. Klibanov, Acc. Chem. Res., 23, 114(1990).
2. b) C.-H. Wong, and J. M. Whitesides, Enzymes in synthetic organic chemistry, Pergamon, Boston (1994).
3. a) R. A. Persichetti, N. L. St. Clair, J. P. Griffith, M. A. Navia, and A. L. Margolin, J. Am. Chem. Soc., 117, 2732 (1995).
4. b) A. S. Bommarius, T. A. Hatton, and D. I. C. Wang, J. Am. Chem. Soc., 117, 4515 (1995).
5. c) Y. Okahata and K. Ijiro, Bull. Chem. Soc. Jpn., 65, 2411 (1992).
6. d) M. Goto, N. Kamiya, M. Miyata, and F. Nakashio, Biotechnol. Prog., 10, 263 (1994).
7. a) Y. Inada, M. Furukawa, H. Sasaki, Y. Kodera, M. Hiroto, H. Nishimura, and A. Matsushima, TIBTECH, 13, 86 (1995).
8. b) Z. Yang, A. J. Mesiano, S. Venkatasubramanian, S. H. Gross, J. M. Harris, and A. J. Russell, J. Am. Chem. Soc., 117, 4843 (1995).
9. a) Y. Ito, H. Fujii, and Y. Imanihsi, Biotechnol. Prog., 9, 128 (1993).
10. b) Y. Ito, H. Fujii, and Y. Imanihsi, Biotechnol. Prog., 10, 398 (1994).
11. K. Chen, and F. H. Arnold, Proc. Natl. Acad. Sci., U.S.A., 90, 5618 (1993).
12. Y. Taniyama, Y. Yamamoto, M. Nakao, M. Kikuchi, and M. Ikahara, Biochem. Biophys. Res. Commun., 152, 962 (1988).
13. Primer 5'-GGTGTTGCCCGGGTCGACCCGGTCGAC-3' was used to introduce a SmaI endonuclease site instead of stop codon of human lysozyme in pLY plasmid by site-directed mutagenesis. Four primers 5'-AATTCTCAGTCATCCCGGGGGGCCCACCACCACCAC-3', 5'-CACCACCACCACCACCATAATAGGTCGACCGACCTGTGCTCA-3', 5'-TGGTGGTGGTGGTGGTGGGCCCCCCGGGATGACTGAG-3' and 5'-AGCTTGAGCACAGGTCGGTCGACCT ATTATGGTGGTGGTGG-3' were annealed to form a double strand DNA fragment coding 10 residues of proline and the stop codon. Subsequently, the DNA fragments were annealed in the presence of pUC19 digested with EcoRI and HindIII and were ligated to construct the pPR10 plasmid. The pPR10a plasmid coding 10 residues of proline without the stop codon was constructed similarly. The pPR20 plasmid coding 20 proline residues was constructed by insertion of the proline region in the pPRlOa plasmid into the upstream of pPR10 plasmid. The pPR30 plasmid was also constructed similarly. The synthesis of plasmid was confirmed by electrophoresis. The pLY was digested with SmaI and SalI endonucleases. The pPR10, pPR20 and pPR30 plasmids were digested with Apd-blunting-SalI endonuclease. Subsequently, pLY-polyproline, which is the plasmid DNA coding the lysozyme carrying polyproline, was constructed by the ligation reaction. Lysozymes carrying various lengths of polyproline at the carboxyl end were expressed in Saccharomyces cerevisiae AH22 (MATa, his3, leu2) with pLY-pPR10, pLY-pPR20, or pLY-pPR30 plasmid. The synthesis of proteins was confirmed by electrophoresis and mass spectroscopy. The fundamental techniques are described in the following paper. T. Kanai, H. Atomi, K. Umemura, H. Ueno, Y. Teranishi, M. Ueda, and A. Tanaka, Appl. Microbiol Biotechnol, 44, 759 (1996).
14. H. R. Ibrahim, M. Yamada, K. Matsushita, K. Kobayashi, and A. Kato, J. Biol. Chem., 269, 5059 (1994).
15. The suspension of Micrococcus lysodeikticus cells (1 mg/ml) was mixed with 100 μl of lysozyme solution (10 Hg/ml) in 0.1 M potassium phosphate buffer (pH 6.2). The mixture was incubated at 25 °C for 3 min. The decrease in absorbance at 450 nm was measured. The activity of wild-type lysozyme was taken as 100%.
16. 4 cells/ml) was mixed with lysozyme (final concentration, 1.2 μg/ml) in 50 mM phosphate buffer (pH 7.0). The mixture was incubated at 37 °C for 60 min and a diluted portion (50 μl) was picked out and plated onto LB agar plates. After incubation of the plates at 37 °C overnight, the number of colonies was counted. The activity of wild-type lysozyme was taken as 100%.
17. The activity of lysozyme was determined by hydrolysis of p-nitrophenyl-tri-N-acethyl-β-chitotrioside. The substrate (6 mM) was dissolved in 0.5 ml of 40 mM citric acid buffer solution (pH 5.08) containing 20% of dioxane. The solution was incubated at 40°C for 3 min followed by addition of lysozyme solution. The absorbance of p-nitrophenol released was monitored at 405 nm. The activity of wild-type lysozyme was taken as 100%.