Gene Cloning, Expression and Identification of Phosphoglyceric Kinase of <i>Streptococcus suis</i> Serotype 2

doi:10.13523/j.cb.20180303

China Biotechnology

2018, Vol. 38

Issue (3): 16-23 DOI: 10.13523/j.cb.20180303

Orginal Article

Gene Cloning, Expression and Identification of Phosphoglyceric Kinase of Streptococcus suis Serotype 2

Xiao-lu GUO^1,²,Xiu-fang GONG²,Jia-feng CHEN²,Chen-xi DING²,Dan HU²,Xiu-zhen PAN²,Chang-jun WANG^1,^2*(

)

1 School of Life Science and Technology,China Pharmaceutical University,Nanjing 211198, China
2 Department of Epidemiology, Medicinal Research Institute, Nanjing Military Command, Nanjing　210002, China

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Abstract Objective:

To prokaryotically clone and express phosphoglycerate kinase(PGK) from Streptococcus suis serotype 2 and determine enzymatic properties of recombination protein.

Methods:

pgk gene was amplified from the 05ZYH33 genome DNA by PCR and inserted into expression vector pET28a by double digestion. The combined prokaryotic expression plasmid pET28a:pgk was subsequently transformed into E.coli BL21 and induced by IPTG. The induction result was identified by SDS-PAGE and LC-MS/MS, the recombination PGK was then purified by Ni affinity chromatography and used for enzymatic activity measurement.

Results:

PGK protein had a high and soluble expression and displayed a molecular weight about 43kDa in E.coli BL21. The enzymatic activity was then measured by using purified PGK. The enzymatic activity of recombination PGK protein was 75U/ml. The optimum temperature and pH was 25℃ and 7.5, respectively. Kinetic analyses with respect to 3-PGA as substrate gave a K_m of 1.744mmol/L and ATP as substrate gave a K_m of 2.266mmol/L.

Conclusion:

The pgk gene has been successfully expressed by prokaryotic expression system and the purified recombinant PGK protein had a similar enzymatic activity other protein enzymes and showed the best enzymatic activity in optimum condition.

Key words： Streptococcus suis serotype 2 Phosphoglycerate kinase Prokaryotic expression Enzymatic activity

Received: 29 September 2017 Published: 04 April 2018

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	Xiao-lu GUO
	Xiu-fang GONG
	Jia-feng CHEN
	Chen-xi DING
	Dan HU
	Xiu-zhen PAN
	Chang-jun WANG

Cite this article:

Xiao-lu GUO,Xiu-fang GONG,Jia-feng CHEN,Chen-xi DING,Dan HU,Xiu-zhen PAN,Chang-jun WANG. Gene Cloning, Expression and Identification of Phosphoglyceric Kinase of Streptococcus suis Serotype 2. China Biotechnology, 2018, 38(3): 16-23.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180303 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I3/16

Fig.1 Amplification of pgk gene and double digestion of recombinant pET28a:pgk plasmid
(a) Cloning of pgk gene fragment M: 250bp DNA marker; 1: pgk gene fragment(1 197bp) (b) Double digestion of plasmid pET28a:pgk. M: 250bp DNA marker; 1: Undigested plasmid pET28a:pgk; 2: Plasmid pET28a:pgk digested by BamHI and XhoI

Fig.2 Expression analysis of instreested protein by SDS-PAGE
(a) M: Protein Marker ;1: Un-induced E. coli BL21/pET28a; 2: E. coli BL21/pET28a induced with IPTG; 3: Un-induced E.coli BL21/pET28a:pgk; 4: E. coli BL21/pET28a:pgk induced with IPTG; 5: Purified PGK protein (b) M: Protein marker ; 1:Soluble cell lysates induced with IPTG; 2: Inclusion body induced with IPTG

Fig.3 PGK mass spectrometry result

Fig.4 The standard curve of NADH

Fig.5 Time-absorbance curve of PGK

Fig.6 The effect of pH on PGK activity

Fig.7 The effect of temperature on PGK activity

Fig.8 The effect of concentration of the 3-PGA on activity of PGK
(a) Variation of the velocity in function of the substrate concentration (b) Double reciprocal plot of PGK activity as a function of the 3-PGA concentration

Fig.9 The effect of concentration of the ATP on activity of PGK
(a) Variation of the velocity in function of the ATP concentration (b) Double reciprocal plot of PGK activity as a function of the ATP concentration


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