High-level Expression and Characterization of Recombinant Porcine Trypsin and Its R122 Site Mutant in <i>Pichia pastoris</i>

doi:10.13523/j.cb.20180508

China Biotechnology

2018, Vol. 38

Issue (5): 56-65 DOI: 10.13523/j.cb.20180508

High-level Expression and Characterization of Recombinant Porcine Trypsin and Its R122 Site Mutant in Pichia pastoris

Pan-pan ZHANG¹,Yan-ji XU¹,Zhi-ke WANG²,Xiao LIU²,Su-xia LI^1,^*(

)

1 East China University of Science and Technology,State Key Laboratory of Bioreactor Engineering,Shanghai 200237,China
2 Shanghai Yaxin Biotehnology Co. Ltd,Shanghai 201108,China

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Abstract

Objective:Study the effect of R122 residue mutation on the stability of recombinant porcine trypsin (RPT).Methods:RPT,mutants mRPT(R122H) and mRPT(R122H/R73G/R130) were expressed in Pichia pastoris GS115 and further purified. The properties and stabilities of RPT and two mutants was investigated and compared.Results:RPT and its mutants were highly expressed in Pichia pastoris. Relative to RPT, mutant mRPT (R122H) and mRPT (R122H/R73G/R130T) showed higher affinity to substrate BAEE, The K_m values were 18.8μmol/L, 9.0μmol/L and 11.0μmol/L, respectively. Increased stability of mutants to high temperature and alkali were observed. And higher resistance against autolysis were got in the presence and without Ca ²⁺. Conclusion:Pichia pastoris can be used to efficiently express RPT and its mutants.Increased stability under alkaline condition and higher thermal stability and higher anti-self-digestion were got in mutant mRPT (R122H) and mRPT (R122H/R73G/R130T) compared to wild-type RPT, which contribute to the site mutation at the R122.

Key words： Recombinant porcine trypsin Pichia pastoris Mutation mRPT(R122H) mRPT(R122H/R73G/R130T) Property Stability

Received: 11 January 2018 Published: 05 June 2018

ZTFLH:

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Corresponding Authors: Su-xia LI E-mail: lisuxia@ecust.edu.cn

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	Pan-pan ZHANG
	Yan-ji XU
	Zhi-ke WANG
	Xiao LIU
	Su-xia LI

Cite this article:

Pan-pan ZHANG,Yan-ji XU,Zhi-ke WANG,Xiao LIU,Su-xia LI. High-level Expression and Characterization of Recombinant Porcine Trypsin and Its R122 Site Mutant in Pichia pastoris. China Biotechnology, 2018, 38(5): 56-65.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180508 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I5/56

Fig.1 Three dimentional structure of porcine trypsin 3D surface structure model of trypsin (left),and the mutation sites (R73,R122,R130) are marked as red.PT and RT 3D cartoon model comparison(right),and the mutation sites (R73,R122,R130) of RPT are marked as black.The 3D structure of trypsin was obtained from the Protein Data Bank (PDB) and The 3D structure of trypsin was obtained using PyMol Version 0.99

Fig.2 Electrophoresis of traget fragment identified by PCR M:DNA mark; 1: Negative control(pPIC-9K-RPT); 2: pPIC-9K-RPT linearized by SacⅠ; 3: pPIC-9K-mRPT(R122H) linearized by SacⅠ; 4: pPIC-9K-RPT linearized by SacⅠ

Fig.3 SDS-PAGE analysis of expression of recombinant RPT and mutants and the activation of the recombinant trypsinogen M:Protein marker; 1: Recombinant porcine trypsinogen; 2, 3: Mutants mRPT(R122H) and mRPT(R122H/R73G/R130T)

Fig. 4 The records of fermentation and the SDS-PAGE analysis of the expression of mRPT(R122H) (a) Time course profile of mRPT(R122H) in an 10L fermenter (b) The trend chart of OD₆₀₀ during the fermenter (c) The SDS-PAGE analysis of the express of mRPT(R122H) M: Protein mark; 1-7: Samples were induced 12h,16.5h,24h,28.7h,33h,37.5h,48h respectively

Fig.5 The purification of mRPT(R122H) (a) The SDS-PAGE analysis of mRPT(R122H) by CM-FF chromatography 1:The loading sample; M:Protein mark; 2:The flow out part; 3:Balance part; 4-14:Eluting parts (b) The SDS-PAGE analysis of mRPT(R122H) by Hitrap benzamidine chromatography M:Protein mark; 1~6:Eluting parts (c) Elution curve

Fig.6 Determination of K_m of RPT and mutants

Table 1 Kinetic parameters of RPT and mutants mRPT(R122H)、 mRPT(R122H/R73G/R130T)

Fig.7 Optimal pH (a) and Effect of pH on the stability (b) of RPT and mutants

Fig.8 Optimal temperature ofRPT(a), mRPT(R122H)(b) and mRPT(R122H/R73G/R130T)(c)

Fig.9 Thermal stability of RPT(a), mRPT(R122H)(b) and mRPT(R122H/R73G/R130T)(c)

Fig.10 Time course of the decrease of activity of RPT and mutants in the presence of 20mmol/L Ca²⁺ (a) and 1mmol/L EDTA (b)

Fig.11 RP-HPLC analysis on self-digestion of RPT and mutants at pH 8.0, 1mmol/L EDTA,25℃ (a), (b)RPT incubated for 0h and 1h (c),(d) mRPT(R122H) incubated for 0h and 1h (e) and (f) mRPT(R122H/R73G/R130T) incubated for 0h and 1h


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