Among the most useful information obtained from the purified lipase was amino
acid sequence data. The sample was sufficiently pure that unambiguous identifica-
tion of the first 28 amino-terminal residues was possible, with a ‘best guess’ possible
for the amino acid sequence of an additional 12 positions. This information was vital
to subsequent characterization of the cloned lipase gene (see Section 4.5).
4.4 Crystallization and determination
of three-dimensional structure
Just as the development of the purification scheme was being completed, the first
high-resolution three-dimensional structures of lipases appeared (Brady et al., 1990;
Winkler et al., 1990). The availability of pure enzyme fostered a similar attempt to
determine the structure of the Rd lipase. Several large lipase crystals were gratify-
ingly obtained within months. However, none of these proved suitable for the col-
lection of diffraction data. Continued efforts over the course of more than two years
were required to identify crystallization conditions that produced diffracting crystals
(Swenson et al., 1994). Subsequently the structure of the lipase was determined to a
resolution of 2.6 A ̊, the fourth fungal lipase structure to be published (Derewenda et
al., 1994a,b,c).
4.4 Crystallization and determination of three-dimensional structure 73
Figure 2. Isoelectric focusing (IEF) of the purified lipase (Haas et al., 1992). (A) Silver-stained IEF gel.
Lane 1, 1.0lg of trypsinogen standard; lane 2, 0.433lg of IEF standard protein preparation; lane 3, 98 ng
of pure lipase. The isoelectric points of the standard proteins are indicated in the left margin. (B) Detection
and localization of lipolytic activity in the purified lipase preparation by activity stain. An IEF gel iden-
tical to that in Panel A was inverted onto lipase indicator media containing olive oil and rhodamine B,
incubated, and photographed under ultraviolet light. The bright band indicates the site of lipase activity.