Open Access Research article

Production of recombinant human annexin V by fed-batch cultivation

Laura S Marder12, Juleane Lunardi24, Gaby Renard4, Diana C Rostirolla13, Guilherme O Petersen124, José E S Nunes4, Ana Paula D de Souza5, Ana Christina de O Dias4, Jocelei M Chies4, Luiz A Basso123, Diógenes S Santos123 and Cristiano V Bizarro12*

Author Affiliations

1 Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681, 90619-900 Porto Alegre, Brazil

2 Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Porto Alegre 90619-900, Brazil

3 Programa de Pós-Graduação em Medicina e Ciências da Saúde, PUCRS, Porto Alegre 90619-900, Brazil

4 Quatro G Pesquisa & Desenvolvimento, LTDA, Porto Alegre 90619-900, Brazil

5 Instituto de Pesquisas Biomédicas (IPB), Laboratório de Imunologia Molecular, PUCRS, Porto Alegre 90619-900, Brazil

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BMC Biotechnology 2014, 14:33  doi:10.1186/1472-6750-14-33

Published: 27 April 2014

Abstract

Background

Annexin V, a 35.8 kDa intracellular protein, is a Ca+2- dependent phospholipid binding protein with high affinity to phosphatidylserine (PS), which is a well-known hallmark of apoptosis. Annexin V is a sensitive probe for PS exposure upon the cell membrane, and used for detection of apoptotic cells both in vivo and in vitro. Large-scale production of recombinant human annexin V is worth optimization, because of its wide use in nuclear medicine, radiolabeled with 99mTc, for the evaluation of cancer chemotherapy treatments, and its use in identification of apoptotic cells in histologic studies. Here we describe the high-yield production of a tag-free version of human annexin V recombinant protein by linear fed-batch cultivation in a bioreactor.

Results

We cloned the human ANXA5 coding sequence into the pET-30a (+) expression vector and expressed rhANXA5 in batch and fed-batch cultures. Using E. coli BL21 (DE3) in a semi-defined medium at 37°C, pH 7 in fed-batch cultures, we obtained a 45-fold increase in biomass production, respective to shaker cultivations. We developed a single-step protocol for rhANXA5 purification using a strong anion-exchange column (MonoQ HR16/10). Using these procedures, we obtained 28.5 mg of homogeneous, nontagged and biologically functional human annexin V recombinant protein from 3 g wet weight of bacterial cells from bioreactor cultures. The identity and molecular mass of rhANXA5 was confirmed by mass spectrometry. Moreover, the purified rhANXA5 protein was functionally evaluated in a FITC-annexin V binding experiment and the results demonstrated that rhANXA5 detected apoptotic cells similarly to a commercial kit.

Conclusions

We describe a new fed-batch method to produce recombinant human annexin V in large scale, which may expand the commercial utilities for rhANXAV to applications such as in vivo imaging studies.

Keywords:
Recombinant human annexin v; Fed-batch cultivation; Large-scale; Apoptosis detection