Constitutive Expression of Human Goose-type Lysozyme 2 in <i>Pichia pastoris</i> Using the GAP Promoter

doi:10.13523/j.cb.20181007

China Biotechnology

2018, Vol. 38

Issue (10): 55-63 DOI: 10.13523/j.cb.20181007

Orginal Article

Constitutive Expression of Human Goose-type Lysozyme 2 in Pichia pastoris Using the GAP Promoter

Peng HUANG^1,^**,***(

),Li-ping YAN^2,^**,Ning ZHANG³,Jin-lei SHI⁴

1 College of Clinical Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
2 Clinical Laboratory, Affiliated Central Hospital of Qingdao University, Qingdao 266042, China
3 College of Basic Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
4 School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China

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Abstract

This study aimed to achieve the constitutive expression of human goose-type lysozyme 2 (hLysG2) in Pichia pastoris using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and to establish an efficient strategy for the production of recombinant hLysG2 (rhLysG2) on a bench scale. The hLysG2 gene was synthesized according to the codon usage preference of P. pastoris and cloned into pGAPZαA vector. The resulting pGAPZαA-hLysG2 plasmid was linearized and transformed into competent P. pastoris GS115, followed by Geneticin screening. The transformants with higher Geneticin resistance were selected to investigate the constitutive expression of rhLysG2 in P. pastoris. The lytic activity of rhLysG2 in the fermentation broth reached its peak after 60h of cultivation. SDS-PAGE and Western blot analysis showed that rhLysG2 was successfully secreted into the fermentation broth. There were a 23.8% increase in the total lytic activity and a 48h reduction in the cultivation time in comparison with those of the P. pastoris strain integrated with the pPIC9K-hLysG2 plasmid. Using chitin affinity and size-exclusion chromatography, rhLysG2 was purified with a yield of 187.4mg/L of fermentation supernatant, above 99.9% purity and a specific activity of 13 500U/mg under the condition of pH 5.6, 0.1mol/L of Na ⁺, 30℃. In conclusion, rhLysG2 was expressed at high level in P. pastoris by codon optimization and had in vitro bactericidal activity against some pathogenic bacteria, which has laid a solid foundation for its possible future pharmaceutical applications.

Key words： Human goose-type lysozyme 2 Pichia pastoris GAP promoter Constitutive expression Bactericidal activity

Received: 16 April 2018 Published: 09 November 2018

ZTFLH:

R392-33R392.11

Corresponding Authors: Peng HUANG,Li-ping YAN E-mail: huangp_15@sumhs.edu.cn

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	Peng HUANG
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	Jin-lei SHI

Cite this article:

Peng HUANG,Li-ping YAN,Ning ZHANG,Jin-lei SHI. Constitutive Expression of Human Goose-type Lysozyme 2 in Pichia pastoris Using the GAP Promoter. China Biotechnology, 2018, 38(10): 55-63.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181007 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I10/55

Table 1 Primers used in this study

Fig.1 Identification of recombinant expression plasmid

Fig.2 Identification of recombinant expression plasmid (a) Lane M: DNA marker; Lanes 1~5: Amplification products (b) Lane M: DNA marker; Lane 1: The pGAPZαA-hLysG2 plasmid digested by EcoR I and Not I

Fig.3 Identification of positive transformants by PCR Lane M: DNA marker; Lane 1: pGAPZαA-hLysG2; Lanes 2~3, 5~6 and 8: Positive transformants; Lanes 4 and 7: Negative clones

Fig.4 Increment curve of lytic activity in the fermentation supernatant

Fig.5 SDS-PAGE analysis of the fermentation supernatant Lane M: Molecular weight marker; Lanes 1~6: 10μl of the fermentation supernatant taken at 0h, 12h, 24h, 36h, 48h and 60h

Fig.6 Elution profile of chitin affinity chromatography

Fig.7 SDS-PAGE (upper) and Western blot analysis (bottom) of purified rhLysG2 Lane M: Molecular weight marker; Lanes 1~4: Purified rhLysG2

Fig.8 HPLC analysis of purified rhLysG2

Table 2 Results of the purification of rhLysG2

Fig.9 Effect of pH, ion concentration and temperature on rhLysG2 activity and stability (a) Activities of rhLysG2 at different pH (b) Effect of ion concentration on rhLysG2 activity (c) Effect of temperature on rhLysG2 activity (d) Effect of temperature on the stability of rhLysG2 All values were normalized to the peak activity for each curve (100%)

Table 3 Effect of metal ions on rhLysG2 activity

Fig.10 Bactericidal activity analysis of rhLysG2 ^** P< 0.01 vs the control group; ^* P< 0.05 vs the control group


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