Introduction
As bioactive molecules, peptides have great application potential in various industrial sectors. However, due to the cost-intensive synthesis and purification, peptides are currently used almost exclusively in medicine. Cost-effective biotechnological production and processing would expand the market potential and fields of application for peptides.
Goals
Therefore, we want to develop a purification method for heterologously expressed peptides utilizing the cellulose binding domain CBD from Trichoderma reesei (T. reesei) in combination with an acid cleavage site with favorable cellulose particles.
Materials and methods
For the biotechnological production we are using the Ascomycete Aspergillus oryzae as expression host. After genomic integration of the modified expression cassette this organism expresses the heterologous peptides combined with a cellulose binding domain (CBD) into the culture medium. After cell separation the cultivation broth is incubated with microcrystalline cellulose and the CBD-tagged peptides are adsorbed from the complex nutrient medium in the presence of 1 M (NH4)2SO4. Separation from the medium is achieved via centrifugation and washed several times with diluted (NH4)2SO4 solution. Even washing with water did not result in CBD detachment from the cellulose indicating a very strong interaction between cellulose and the CBD. To guarantee the purification as well as to remove the peptide attached CBD we cloned an acid cleavage site in between. Finally, the detachment of the peptides from the CBD bound to the cellulose is done by chemical acid cleavage and the purified peptides are separated from the cellulose via centrifugation or filtration.
Summary
Using the CBD from T. reesei combined with an acid cleavage site attached to heterologously expressed peptides we developed a very cost-effective peptide isolation technique which can expand the fields of application of peptides in the future.