Midkine is a heparin-binding growth factor that promotes cell growth, survival, and migration. Externally
added midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction in the mouse.
Preclinical testing of this protein is in progress. Externally added pleiotrophin, a member of the midkine protein family,
promotes functional recovery after neural transplantation in rats. Thus, pleiotrophin is also a candidate therapeutic protein.
Large amounts of these proteins were obtained by using the heterologous protein expression system of Pichia pastoris,
and the recombinant P. pastoris clones were cultured in a controlled fermentor. Intracellular expression yielded about 300
mg/L recombinant human (rh)-midkine, which was extracted, renatured, and purified. From 1 L of the culture, 64 mg of
rh-midkine was purified. Secretory expression induced by the midkine secretion signal resulted in about 100 mg of rhmidkine
in 1 L of the culture supernatant, but over 70% of the rh-midkine had yeast-specific glycosylation. Three threonyl
residues that are targets for glycosylation were substituted with alanyl residues, and nonglycosylated, active rh-midkine
was obtained. In secretory expression using α-mating factor prepro-sequence, about 640 mg/L rh-midkine was obtained,
but it was partially truncated. Therefore, a protease-deficient host was used, and about 360 mg/L intact rh-midkine was
then obtained. The rh-midkine was recovered and purified, with 70% final yield. All purified rh-midkine, regardless of
expression method, was able to promote mammalian cell proliferation. In secretory expression of rh-pleiotrophin using α-
mating factor prepro-sequence, 260 mg/L rh-pleiotrophin could be secreted. The rh-pleiotrophin was recovered and efficiently
purified with 72% final yield.