rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model

doi:10.5246/jcps.2014.01.003

Journal of Chinese Pharmaceutical Sciences ›› 2014, Vol. 23 ›› Issue (1): 22-27.DOI: 10.5246/jcps.2014.01.003

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rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model

Yanan Zhu, Yuanjun Zhu, Qixin Bu, Xiaoyan Liu, Yinye Wang^*

Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2013-04-24 Revised:2013-05-10 Online:2014-01-23 Published:2014-01-22
Contact: Yinye Wang*
About author:*Corresponding author. Tel.: 86-10-82802652; Fax: 86-10-62015584; E-mail: wangyinye@bjmu.edu.cn
Supported by:
National Technology Graveness Special Purpose Fund (Grant No. 2009ZX09301-010).

Abstract

Abstract:

Recombinant ancylostoma caninum anticoagulant peptide-5 (rAcAP5) has been reported to inhibit thrombin-activatable fibrinolysis inhibitor (TAFIa) activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion (MCAO) model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow (rCBF) was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB1-rAcAP5. AcAP5 (28 mg) with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle (normal saline) treatment did not change the rCBF, while treatment with rAcAP5 (50–200 µg/kg, i.v.) increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.

Key words: rAcAP5, Over-expression, Purification, Thrombolysis

CLC Number:

R965

Supporting:

Yanan Zhu, Yuanjun Zhu, Qixin Bu, Xiaoyan Liu, Yinye Wang*. rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model [J]. Journal of Chinese Pharmaceutical Sciences, 2014, 23(1): 22-27.

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rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model

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