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 l -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.

Analysis of Recombinant Human Serum Albumin Extraction and Degradation in Transgenic Rice Extracts

“…The resulting rHSA concentrations at 10 min (2.0 mg/g flour) and in the clarified extract (2.1 mg/g flour) are similar to the rHSA concentration of pH 3.5 at room temperature at 10 min (2.2 mg/g flour). Because lower temperature typically reduces protein extractability, the amount of TSP and rHSA was evaluated and compared to that at room temperature.…”

“…The effect of temperature on extraction is another parameter to consider as recombinant human proinsulin extraction from transgenic corn showed a positive effect with temperature increase. The protein concentrations achieved for extraction performed at 25°C (18.87 ± 0.48 mg/L) were 10% less than at a higher temperature of 40°C (20.72 ± 1.06 mg/L) . Other protein extraction studies with transgenic corn show increased solubility with an increase of temperature.…”

Compendium on Food Crop Plants as a Platform for Pharmaceutical Protein Production

“…Human insulin was one of the first pharmaceutical proteins that was produced in bacteria cells using recDNA technology. Over time, it transpired that fully functional insulin can be successfully produced in the seeds of a plant (e.g., A. thaliana , Zea mays ) [ 116 , 124 ]. Using the oleosin fusion strategy, the accumulation of the described protein was achieved at the level of over 0.1% of TSP (total soluble protein) [ 124 ].…”

Plant Platforms for Efficient Heterologous Protein Production

“…Numerous antigens have been expressed in corn seeds owing to high biomass yield, bigger endosperm, absence of active proteases in dry seeds and presence of a rich mix of molecular chaperones and disulfide isomerases for proper folding of proteins and the very well-established processing technology [117,118]. Heat labile B subunit from enterotoxigenic E. coli, cholera toxin B subunit from V. cholerae, spike protein of swine transmissible gastroenteritis, hepatitis B oral vaccine and human recombinant proinsulin was efficiently expressed in corn plants [119][120][121][122][123][124].…”