Recombinant Expression of Human <em>LYC5</em> Lysozyme in <em>Pichia pastoris</em>

doi:10.13523/j.cb.20140401

China Biotechnology

2014, Vol. 34

Issue (4): 1-8 DOI: 10.13523/j.cb.20140401

Recombinant Expression of Human LYC5 Lysozyme in Pichia pastoris

WEI Tian-tian, YU Ying, JIN Xiao-feng, TAO Jian-jun, YU Long

State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China

Download:

HTML

PDF(1185KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

LYC5 is a kind of c type human lysozyme protein. To optimized and designed the LYC5 gene based on the coding sequence of mRNA on GenBank and the preference of Pichia yeast codon, then cloned the encoding gene into the expression plasmid pPIC9K,and the resulting recombinant plasmids were named as pPIC9K- LYC5. Then they were linearized and electro-transformed into the Pichia pastorios GS115 express bacterium,the multiple copies of transformants were screened by Minimal Dextrose medium (MD),Minimal Methanol medium (MM),the antibiotic of G418 and PCR identification. The transformants were screened through culturing and induction under methanol in baffled flask,the results of SDS-PAGE indicated that the LYC5 target proteins were secreted into the supernatant, about 15 kDa in sizes. These proteins were identified as LYC5 lysozymes by LTQ orbitrap elite MS analysis,with expression levels being about 20 mg/L. The expression products had obvious antibacterial activity on Micrococcus lysodeikticus,with the activity was about 40 000 U/mg,the optimum temperature and pH for reaction of LYC5 lysozyme were 45℃ and 5.0. The successfully expressed LYC5 lysozyme protein with antibacterial activities,which laid a foundation for deeping discuss the antimicrobial spectrum and application of human lysozyme family.

Key words： Human lysozyme LYC5 Pichia pastorios pPIC9K

Received: 03 March 2014 Published: 25 April 2014

ZTFLH:

Q786

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors

Cite this article:

WEI Tian-tian, YU Ying, JIN Xiao-feng, TAO Jian-jun, YU Long. Recombinant Expression of Human LYC5 Lysozyme in Pichia pastoris. China Biotechnology, 2014, 34(4): 1-8.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20140401 OR https://manu60.magtech.com.cn/biotech/Y2014/V34/I4/1

[1] Gao Yu,Zhao Hong Lei,Feng Xin, etal.Expression of recombinant human lysozyme-tachyplesin 1(hLYZ-TP1) in Pichia pastoris and analysis of antibacterial activity.Biomed Environ Sci,2013,26(4):319-322.

[2] Tetsuya Masuda,Yuki Ueno, Naofumi Kitabatake.High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris.Protein Expression and Purification,2005,39:35-42.

[3] Lien Callewaert, Chris W Michiels.Lysozymes in the animal kingdom.J Biosci,2010,35:127-160.

[4] Lan-ping Yu,Bo-guang Sun,Jun Li,etal.Characterization of a c-type lysozyme of Scophthalmus maximus:Expression,activity,and antibacteral effect.Fish & Shellfish Immunology,2013,34:46-54.

[5] Yang B, Wang J, Tang B, etal.Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.PLoS ONE,2011,6(3):e17593.

[6] Jianmin Huang,Liying Wu,Dorice Yalda,etal.Expression of functional recombinant human lysozyme in transgenic rice cell culture.Transgenic Research,2002,11:229-239.

[7] SylviaLee-Huang,Paul L Huang,YongtaoSun,et al.Lysozyme and Rnases as anti-HIV components in β-core preparations of human chorionic gonadotropin.Proc Natl Acad Sci,1999,96:2678-2681.

[8] Chen Yao-Tseng,Christian Iseli,Charis A Venditti,et al.Identification of a new cancer/testis gene family,CT47,among expressed multicopy genes on the human X chromosome.Genes, Chromosomes & Cancer,2006,45:392-400.

[9] 张克雄. 人体 LYC4 基因克隆暨 LYC4、LYC3、Cdv-1R 与生殖关系的初步研究. 上海: 复旦大学,2004. Zhang K X.Gene cloning of human LYC4 cum a preliminary study of the relationship among LYC4、LYC3、Cdv-1R and reproduction.Shanghai:Fudan University, 2004.

[10] 黄鹏. 人c型和g2型溶菌酶的功能研究及人c型溶菌酶制备条件的优化. 上海: 复旦大学,2008. Huang P.Function of human c-type and g2-type lysozyme and optimization of the preparation conditions of human c-type lysozyme.Shanghai:Fudan University,2008.

[11] Kenji Maehashi,Mami Matano,Tomohiro Irisawa,et al.Molecular characterization of goose- and chicken-type lysozyme in emu(Dromaiusnovae-hollandiae):evidence for extremely low lysozyme levels in emu egg white.Gene,2012,492:244-249.

[12] Brigitte Gasser,Roland Prielhofer,Hans Marx,et.al.Pichia pastoris:protein production host and model organismfor biomedical research.Future Microbiol,2013,8(2):191-208.

[13] Schmidt F R.Recombinant expression systems in the pharmaceutical industry.Appl Microbiol Biotechnol,2004,65:363-372.

[14] Goncalves A M, Pedro A Q,Maia C,et.al.Pichia pastoris:A recombinant microfactory for antibodies and human membrane proteins.J Microbiol Biotechnol,2013,23(5):587-601.

[15] James M Cregg,Joan Lin Cereghino,Shi Jian-ying,et al.Recombinant protein expression in Pichia pastoris.Molecular Biotechnology,2000,16(1): 23- 52.

[16] Klockars M, Reitamo S.Tissue distribution of lysozyme in man.J Histochem Cytochem,1975,23: 932-940.

[17] Mason D Y, Taylor C R.The distribution of muramidase(lysozyme).J Clin Path,1975,28:124-132.

[1]	HUANG Peng, LI Wen-shu, XIE Jun, BAO Jian-ying, CAO Xiao-e, YU Long, XU Yi-xin . *Expression of Human Lysozyme-like Protein 6 in Pichia pastoris* and Analysis of Enzymatic Activity of the Protein**[J]. China Biotechnology, 2015, 35(8): 30-37.

[2]	LI Xin-xin, TAO Jian-jun, YU Long. *Recombinant Human Lysozyme LYZL4：Expression in Pichia pastoris* and Its Antibacterial Activity**[J]. China Biotechnology, 2014, 34(1): 79-85.

[3]	MING Fei-ping, YANG Jun, ZHU Jin-mei, KUANG Zhe-shi, LI Hua-zhou, XIA Feng-geng, YE Ming-qiang, WANG Hou-guang, ZHAO Xiang-jie, HUANG Zhi-feng, MA Miao-peng, SHI Ju-qing, CAI Hai-ming, ZHANG Ling-hua. Modification of 5’UTR Sequences of pPIC9K Increases Expression of Antimicrobial Peptide PR39[J]. China Biotechnology, 2013, 33(12): 86-91.

Viewed

Full text

Abstract

Cited

Shared

Discussed