Purification and characterisation of cell survival factor 1 (TCSF1) from Tetrahymena thermophila

Abstract

Of a number of peptides isolated from the extracellular medium of Tetrahymena cultures, two with masses 9.9 and 22.4 kDa allowed low-density cultures of this ciliate to survive and enter a proliferate phase. The smaller peptide (TCSF1) also greatly helped cultured mammalian fibroblasts to survive in medium containing very low concentrations of serum for considerably longer than controls, and to grow when full strength medium was restored. The primary sequence of the TCSF1 was determined, and synthetic TCSF1 was observed to exhibit rescuing activity comparable to that of the native peptide.

Fig. 1

Survival of T. thermophila cultures from 100 cells per ml supplemented with HPLC fractions, the x-axis relating the fraction to the strength of acetonitrile used in the preparation prior to lyophilisation. The y-axis gives the number of divisions (generations) the cells accomplished in 24 h

Fig. 2

Conditioned medium separated on native 18% gel. The 15 clearest bands were cut from the gel and the proteins were extracted

Fig. 3

a, b Conditioned CDM was separated on 18% native gels. Protein was extracted from the bands and used as a supplement to fresh medium. Cells inoculated into the test media divided more in 24 h as compared to the controls, when supplemented with protein from bands 2, 7–8 and 12. An inhibitory effect was observed when using supplements from bands 1, 6 and 14. Figure shows average values for three cultures supplemented with each band. Error bars are 1 SD; t test for “crude”, 8, 9, 12, 13 and 14 gives their p values

Fig. 4

Rescuing activity of TCSF1 synthesized from the HPLC G sequence. Synthetic TCSF1 rescued cells seeded a sub-critical density in all cases at concentrations higher than 1 μM

Fig. 5

Early proliferation in high-density culture (inoculated with 177 cells/ml in CDM) of T. thermophila with synthetic TCSF1 supplement (filled square) or without (open square). Mean ± 1 SD

Fig. 6

Thymidine incorporation in T. thermophila cultures f similar density to those shown in Fig. 5, supplemented with native TCSF1 (filled square), synthetic TCSF1 (filled circle); or neither as the control (open square). Mean ± 1 SD

Fig. 7

The current three-dimensional model of TCS1 as a dimer—the colours are self-explanatory for a peptide and the two lines indicate possible disulphide bridges between the monomers