Recombinant human proinsulin from transgenic corn endosperm: Solvent screening and extraction studies

Abstract

Recombinant pharmaceutical proteins are being produced in different systems such as bacteria and mammalian cell cultures. The use of transgenic plants as bioreactors has recently arisen as an alternative system offering many practical and economic advantages. However, finding an optimum strategy for the downstream processing (DSP) of recombinant proteins from plants still remains a challenge. In this work, we studied the extraction of recombinant human proinsulin (rhProinsulin) produced in the endosperm of transgenic corn seeds. An efficient extraction solvent was selected and the effects of temperature, solvent-tosolid ratio, time, and impeller rotational speed on the extraction were evaluated using an experimental design. After an extraction kinetics study, temperature was further evaluated to maximize rhProinsulin concentration in the extracts and to minimize the native corn components carbohydrates, phenolic compounds, and proteins. A high efficiency condition for extracting rhProinsulin with the selected solvent - 50 mM sodium bicarbonate buffer pH 10.0 and 5 mM DTT - was an extraction time of 2 h at a solvent-to-solid ratio of 10:1 and 25°C. The maximum rhProinsulin concentration in the extracts at that condition was 18.87 mg 1-1 or 0.42% of the total soluble protein. These values are within the range in which the production of pharmaceutical proteins in plants can be competitive with other expression systems. The results presented provide information for the development of an additional production platform for the hormone insulin.