Production of recombinant human annexin V by fed-batch cultivation

Abstract

Doc number: 33

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 ^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.