To improve the expression of equistatin, a proteinase inhibitor from the sea anemone Actinia equina, in the yeast Pichia pastoris, we prepared gene variants with yeast-preferred codon usage and lower repetitive AT and GC content. The full gene optimization approximately doubled the level of steady-state mRNA and protein accumulated in the culture medium. The removal of a short stretch of 12 additional nucleotides from the multiple cloning site (MCS) sequence in the vector pPIC9 had an enhancement effect similar to full gene optimization (factor 1.5) at the mRNA level. However, at the protein level, this increase was 4- to 10-fold. The optimized gene without the MCS sequence yielded 1.66 g/L active protein in a bioreactor and was purified by a new two-step procedure with a recovery of activity that was >95%. This production level constitutes an overall improvement of about 20-fold relative to our previously published results. The characteristics of the MCS sequence element are discussed in the light of its apparent ability to act as negative expression regulator.