ASMS 2009 poster

Figure 4. Venn diagram showing the intersection of the proteins secreted by

different strains of T. thermophila (Tt1, Tt2 and Tt3) when cultured under starvation

conditions and detected by mass spectrometry. Data is highlighted further in Table 1.

Effects of Strain Type and Growth Conditions on the Secretome of Tetrahymena thermophila CL Madinger1; K Collins2; CH Taron1; JS Benner1

1New England Biolabs Inc., Ipswich, MA; 2University of California Berkeley, Berkeley, CA

Introduction Tetrahymena thermophila, a ciliated protozoa, is inexpensive to culture and can be

grown to high cell densities. While T. thermophila appears to be an excellent

candidate for use in heterologous protein expression, it is essential to know what

proteins are intrinsically secreted. Some proteins within the secretome may be

detrimental to heterologous protein expression. Detrimental proteins of T.

thermophila could be genetically modified to suppress their expression and therefore

optimize protein expression. It is possible that some detrimental protein expression

can be controlled by strain type and/or growth condition, therefore rendering genetic

manipulation unnecessary. Identification of proteins intrinsic to its secretome is the

first step in the optimization of T. thermophila for its use in heterologous secreted

protein expression.

Methods Strains and Culture Conditions

Tetrahymena thermophila (Figure 1) strains Tt1, Tt2 and Tt3 were cultured in flasks

at 30°C with 150 rpm shaking. Cells were cultured in Neff media (0.25% proteose

peptone, 0.25% yeast extract, 0.5% glucose, 3.3 μM FeCl3) and the samples were

divided when cells were in log phase of population growth (~2x105/mL). Half of the

culture continued growing overnight into stationary phase (~1x106/mL) while the

other half was harvested and transferred to starvation media (10 mM Tris-HCl, pH

7.5) for starvation overnight.

For the protein secretion analysis, cells were harvested in a tabletop Eppendorf

centrifuge at no more than 4000 rpm. The spent culture media was filtered (acrodisc

0.2 micron low protein retention syringe filter) and concentrated (20 to 50-fold) by

Vivaspin centrifugation (10 kDa cut-off). These samples were then aliquotted for

analysis by SDS-PAGE and MS.

For the whole cell analysis, cells from T. thermophila strain Tt2 were pelleted from

overnight growth in Neff media and frozen at -80°C. The pellet was resuspended in

50 mM Tris, pH 8, 0.1% SDS and boiled for 3 minutes at 97°C. The sample was

then brought to 0.1% TFA and spun to remove cellular debris.

Multi-dimensional Protein/Peptide Separation

Proteins from the T. thermophila whole cell sample and T. thermophila Tt1, Tt2 and

Tt3 samples grown under vegetative conditions were separated by HP-RPLC via a

PLRP-S column. Fractions were collected, dried to completion, resuspended in

reaction buffer and digested overnight with TPCK-treated Trypsin (New England

Biolabs). Peptides were then separated by an integrated C18 trap/column/needle and

analyzed online by nanoESI-MS/MS with an Ion Trap Mass Spectrometer (Agilent

Technologies) and ChipCube. These analyses were performed in triplicate.

Single-dimensional Peptide Separation

Proteins from the T. thermophila Tt1, Tt2 and Tt3 strains grown under starvation

conditions were dried to completion, resuspended in reaction buffer and digested

overnight with TPCK-treated Trypsin (New England Biolabs). Peptides were

separated by an integrated C18 trap/column/needle and analyzed online by nanoESI-

MS/MS with an Ion Trap Mass Spectrometer (Agilent Technologies) and ChipCube.

These analyses were performed in triplicate.

Protein Identification using MS and MS/MS Data

The MS/MS data were analyzed using Mascot (Matrix Science) and Spectrum Mill

(Agilent Technologies). Peptides generated by a tryptic digest were searched against

the T. thermophila genome in a T. thermophila database that was generated by

combining the T. thermophila genome with Swiss-Prot database (Version 51.6).

Proteins scoring greater than 67 and 20 were considered valid identifications (for

Mascot and Spectrum Mill, respectively) and were combined to generate the final

list of identified proteins.

Overview • Three strains of Tetrahymena thermophila (Tt1, Tt2 and Tt3) were cultured under

two conditions (rich media and starvation) and secreted proteins were identified by

online ESI-MS/MS.

• Growth conditions affect the protein composition of the T. thermophila secretome.

Proteins in common between different conditions are called the “base secretome”.

• Additionally, whole cell proteins of Tt2 were identified by 2D-LC MS/MS.

• There were 43 proteins in common between the Tetrahymena thermophila

secretomes (6 conditions) and whole cell extract.

Results

• In total, 492 proteins were identified from the analysis of Tetrahymena thermophila cultured under multiple conditions and highly expressed in

whole cells. There are a total of 24,725 proteins in the predicted proteome [2].

• A secreted compliment of 247 proteins were identified from the analysis of Tetrahymena thermophila cultured under six different conditions:

strains Tt1, Tt2 and Tt3 cultured under both rich media and starvation conditions.

• We identified 288 proteins from our analysis of T. thermophila whole cells, which is similar to the number previously reported (223) for cilia

proteins [3]. Of the 288 proteins identified, only 43 total proteins were detected in the combined secretomes of T. thermophila cultured under

six different conditions (data not shown). Considering strain-specific results, only 1 protein was in common between the whole cell proteins

and secreted proteins from Tt1 cultured under rich media and starvation conditions (Figure 3).

• Far fewer proteins were secreted by T. thermophila strains Tt1, Tt2 and Tt3 when cultured under starvation conditions versus rich media

(Figures 4 & 5). Additionally, the base secretome of T. thermophila strains Tt1, Tt2 and Tt3 was much smaller when cultured under starvation

conditions (11 proteins; Figure 4) than when cultured with rich media (33 proteins; Figure 5).

• There is little overlap in the secreted proteins detected between starvation and rich media conditions for the T. thermophila strains Tt1, Tt2, and

Tt3 (Figures 6A, B, and C, respectively). For instance, 40 secreted proteins were detected by MS when strain Tt3 was cultured under starvation

conditions whereas 65 proteins were detected with rich media: of these proteins, only 17 were detected under both conditions (Figure 6C).

Conclusions The Tetrahymena thermophila secretomes from multiple strains and culture conditions were examined by both 1D- and 2D-LC MS/MS

analyses. Additionally, we used 2D-LC MS/MS to examine the proteins expressed at high levels in whole cells of Tetrahymena thermophila.

We have had success using this method in previous secretome analyses [4,5] and the 2D separation was essential for our analyses here because

of the additional complexity present with Tetrahymena samples.

The overlap observed between the secretome and whole cell proteins suggests that the proteins detected are a result of secretion rather than

cell lysis. In examining the individual data sets, there were several unexpected results. First, more proteins characterized as being part of

protein synthesis appear to be expressed during starvation conditions. Second, the percentage of proteases detected stays relatively constant

regardless of strain or growth condition. And finally, the high number of unknown proteins identified indicates that further elucidation is

required before our results can be completely interpreted.

References

1. Image courtesy of Brady Culver, University of Colorado.

2. Coyne RS, Thiagarajan M, Jones KM, Wortman JR, Tallon LJ, Haas BJ, Cassidy-Hanley DM, Wiley

EA, Smith JJ, Collins K, Lee SR, Couvillion MT, Liu Y, Garg J, Pearlman RE, Hamilton EP, Orias

E, Eisen JA, Methé BA. (2008) Refined annotation and assembly of the Tetrahymena thermophila

genome sequence through EST analysis, comparative genomic hybridization, and targeted gap

closure. BMC Genomics. 2008 Nov 26;9:562.

3. Smith JC, Northey JG, Garg J, Pearlman RE, Siu KW. (2005) Robust method for proteome analysis

by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena

thermophila. J Proteome Res. May-Jun;4(3):909-19.

4. Swaim CL, Anton BP, Sharma SS, Taron CH, Benner JS. (2008) Physical and computational

analysis of the yeast Kluyveromyces lactis secreted proteome. Proteomics Jul;8(13):2714-23.

5. Madinger CL, Sharma SS, Anton BP, Fields LG, Cushing ML, Canovas J, Taron CH, Benner JS.

The Effect of Carbon Source on the Secretome of Kluyveromyces lactis. In press.

Figure 2. Analysis of the supernatants of different T. thermophila strains (Tt1, Tt2 and

Tt3) cultured under two conditions (rich media and starvation) by sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE). Lane 5 (Std.) is New England Biolabs

protein marker, the bands corresponding to 27 and 66 kDa are most intense. Lanes 1

through 4 contain samples grown with rich media: Lane 1 is media (M), Lanes 2, 3 and 4

are strains Tt1, Tt3 and Tt2, respectively. Lanes 6 through 9 contain samples that were

starved: Lane 6 is media (M), Lanes 7, 8 and 9 are strains Tt1, Tt3 and Tt2, respectively.

Figure 5. Venn diagram showing the intersection of the proteins secreted by different

strains of T. thermophila (Tt1, Tt2 and Tt3) when cultured with rich media and

detected by mass spectrometry. Data is highlighted further in Table 2.

Table 1. T. thermophila base secretome proteins identified when cultured under

starvation conditions

Figure 1. Picture of Tetrahymena thermophila [1]. Wild type

cells labeled with an anti Cen1p antibody and the 12G10 (Atu1p)

monoclonal antibody from Joe Frankel. Cen1 is red and labels basal

bodies, Atu1 is green and labels microtubules, and DAPI is blue and

labels DNA.

TTHERM_00079450 Mitochondrial glycoprotein

TTHERM_00079600 Papain family cysteine protease

TTHERM_00216010 FTT18; 14-3-3 protein

TTHERM_00378890 GRL5; calcium-binding protein of dense core granules

TTHERM_00402120 Glutathione S-transferase domain containing; eEF-1

TTHERM_00474970 60s acidic ribosomal protein

TTHERM_00522940 Hypothetical; starvation/conjugation ESTs support the prediction

TTHERM_00661740 Hypothetical; one of familyA of closely related hypothetical proteins

TTHERM_00881440 Papain family cysteine protease/CYP1; starvation-induced cysteine protease

TTHERM_00938820 eEF2, translation elongation factor 2

TTHERM_01018540 GRL9; granule lattice protein, dense core granules

TTHERM_00066890 Peptidase C13 family protein

TTHERM_00079450 Mitochondrial glycoprotein



TTHERM_00086780 CCF12; C-terminal crystallin fold protein, dense core granules

TTHERM_00094060 Hypothetical


TTHERM_00268060 Papain family cysteine protease/CysP5


TTHERM_00442170 Hypothetical; chitinase active site glycoside hydrolase domain

TTHERM_00515220 Hypothetical; chitinase active site glycoside hydrolase domain



TTHERM_00590090 CMB1/p85; calmodulin-binding protein; roles in cytokinesis and phagocytosis

TTHERM_00606960 SerH3 immobilization antigen; GPI-linked cell surface antigen

TTHERM_00641150 Papain family cysteine protease; microarray 11th top constitutive







TTHERM_00683010

PLA1/CysP3; Lysosomal phospholipase A1; "may be secreted into the environment as part

of an attack and defense system,"



TTHERM_00760310 Papain family cysteine protease/tetrain/CysP6

TTHERM_00881440 Papain family cysteine protease/CYP1; starvation-induced cysteine protease


TTHERM_00951910 Hypothetical; Glycoside hydrolase, chitinase active site domain

TTHERM_00951920 Hypothetical; Glycoside hydrolase, chitinase active site domain


TTHERM_00662749

(373600685) Hypothetical

TTHERM_00102779

(704849)

cathepsin z; 70% similarity to TTHERM_00102770 Papain family cysteine protease

containing protein

Table 2. T. thermophila base secretome proteins identified when cultured with rich media

Discussion

• Since there was minimal overlap between the secreted and whole cell proteins detected (Figure 3), we conclude that cell lysis is a negligible

contribution to proteins detected in the “secretome” of Tt1, Tt2 and Tt3.

• Growth condition (rich media versus starvation) affected T. thermophila protein expression both quantitatively (Figure 2) and qualitatively

(Figure 7). For instance, the functional groupings and the populations of these groups were both affected when strain Tt2 was cultured under

rich media and starvation conditions (Figure 7B). This variance was also observed when comparing the three strains cultured under the

starvation condition (Figure 7A). Therefore we cannot say whether variance in strain or culture condition had a greater qualitative effect on

protein expression.

• Regardless of strain or culture conditions, proteases always comprise the largest group of proteins identified to be expressed (<25%, Figure 7).

Since this was not a quantitative study, we can not presently determine whether just a broad spectrum of proteases are secreted or what the

variation in the levels of secreted proteases are. Due to the high number of proteases expressed under all culture conditions and in all strains no

one condition examined may be ideal for general protein expression.

• The consistently second largest functional grouping of secreted proteins remain functionally uncharacterized (Figure 7). The elucidation of the

function of the uncharacterized proteins of T. thermophila is important for a full interpretation of our results.

• T. thermophila grown under certain conditions secrete a mucus or slime. This mucus makes direct shotgun MS/MS analysis difficult or

impossible. Our 2D-LC methodology separates the mucus from the protein and no further interference with MS/MS data collection occurs.

Figure 3. Venn diagram showing the intersection of the proteins secreted by

T. thermophila strain Tt1 whole cell and strain Tt1 secreted proteins cultured with

rich media and starvation conditions.

Acknowledgments The authors would like to thank

New England Biolabs and Don

Comb for their support. We also

thank Lauren Fields and Anne-

Lise Fabre for their technical

assistance.

Figure 6. Venn diagram showing the intersection of proteins secreted when

cultured under two conditions (rich media and starvation) for T. thermophila

strains A) Tt1; B) Tt2; and C) Tt3.

A.

B.

C.

M Tt1 Tt3 Tt2 Std. M Tt1 Tt3 Tt2

kDa

212

116

66

43

27

14

Rich Media Starvation

Protease

Secretory Transport

Signaling

Unknown

Structural

Protein Synthesis and Folding

Glycolytic Enzyme

Glycosyl Hydrolase

Stress Response

Defense

Other

Figure 7. Distribution of the detected T. thermophila

secretome proteins according to their different GO

terms: A) Tt1 (outer circle), Tt2 (middle circle) and Tt3

(inner circle) cultured under starvation conditions; B)

Tt2 cultured under rich media (outer circle) and

starvation (inner circle) B.

A.

Tt1 Tt2

Tt3

Rich Media Secretome

Starved Media Secretome

Education

ASMS 2009 poster