We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the Delta snf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the Delta snf2 disruptant. To analyze the expression of Dga1p in wild type and the Delta snf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the Delta snf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the Delta snf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the Delta snf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1 Delta N2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1 Delta N2p is activated Dga1p. Dga1 Delta N2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.