Purification and properties of a factor from insect hemolymph that promotes multicellular vesicle formation in vitro

Abstract

AbstractA vesicle promoting factor (VPF) in an insect cell line IAL‐TND1 was partially purified from larval hemolymph of the cabbage looper, Trichoplusia ni Hübner. The polypeptide had a molecular weight estimated to be between 20.5 kD and 37.5 kD by gel permeation, 22.5 kD by the Ferguson plot on nondenaturing polyacrylamide gel electrophoresis, and 16.88 kD on sodium dodecyl sulfate PAGE. VPF fractions were isolated from hemolymph by gel permeation on Fractogel® and were either subjected to chromatofocusing or preparative isoelectric focusing. After the gel permeation step, the VPF polypeptide was highly unstable during the separation and storage. The two active fractions from the isoelectric separations had isoelectric points of 6.21 and 6.36 and had specific activities of 34 and 32 vesicles/μg protein per test culture chamber. The percentage of total larval equivalent hemolymph proteins found in these fractions was less than 1%. Chromatofocusing technique also yielded an active fraction containing a single band on nondissociating electrophoresis that had VPF activity. This band had an isoelectric point of 6.60 but had a lower specific activity of three vesicles/μg protein in the VPF cell bioassay.