Production of recombinant human annexin V by fed-batch cultivation.
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 (99m)Tc, 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.
Laura S
LS
Marder
Juleane
J
Lunardi
Gaby
G
Renard
Diana C
DC
Rostirolla
Guilherme O
GO
Petersen
José E S
JE
Nunes
Ana Paula D
AP
de Souza
Ana Christina
AC
de O Dias
Jocelei M
JM
Chies
Luiz A
LA
Basso
Diógenes S
DS
Santos
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. cristiano.bizarro@pucrs.br.
Cristiano V
CV
Bizarro
D017304:Annexin A5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D018533:Biomass; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D017304:Annexin A5; D011994:Recombinant Proteins; D016650:Fluorescein-5-isothiocyanate