Development of the Fundamentals of a Technology for the Production of Tribolium castaneum Recombinant Cathepsin L in Komagataella kurtzmanii Yeast

Abstract

Based on the Komagataella kurtzmanii yeast, a strain producing Tribolium castaneum recombinant procathepsin (rpTcCathL) has been obtained; this strain provided biosynthesis and secretion of at least 0.5 g/L of the target protein as a result of cultivation in flasks on a medium of complex composition. It was determined that the pH of the yeast culture medium (pH ≥ 6.5) is the key factor that influences the biosynthesis and accumulation of procathepsin. A new chromatographic purification scheme was developed, which allows one to obtain recombinant procathepsin with a yield of about 30% and a purity of over 95%. It was shown that complete autocatalytic activation of the proenzyme is achieved within no more than 30 min at a temperature of 37°C and pH 4.0–4.5. The resulting recombinant cathepsin and activated cathepsin at a concentration of 0.5 mg/mL and below could be stored without detectable changes at a temperature of 4°C for at least 2 weeks. Thus, the research made it possible to develop the fundamentals of the technology for obtaining and storing recombinant preparations of the highly purified major digestive cathepsin L from T. castaneum in the form of a proenzyme and mature cathepsin L.