From the Serono Reproductive Biology Institute, Rockland, Massachusetts 02370
Received for publication, November 25, 2002 , and in revised form, April 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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Tumor necrosis factor-
(TNF-
)1 is an
attractive therapeutic target for several diseases including rheumatoid
arthritis and Crohn's disease
(4,
5). TNF-
initiates
pleiotropic cytokine regulating effects by binding specifically to multimeric
forms of TNF receptors at high affinity
(6). Protein therapeutics in
this area are focused on selectively neutralizing the activity of TNF-
with either a monoclonal antibody or a soluble TNF receptor. The soluble
extracellular domain of the human p55 TNF receptor exemplifies a protein
therapeutic under development to control the signaling initiated by
TNF-
. This soluble molecule is responsible not only for inactivation of
the TNF, but also for its clearance
(7,
8). We previously found the
expression level of TNFrED in CHO cells as measured in picograms/cell/day to
be low when compared with other proteins such as immunoglobulins. Analysis of
these transfected CHO cells revealed that the rate-limiting step in the
expression of TNFrED appears to be either at translation or in a
post-translational
process.2 A possible
bottleneck in the expression of TNFrED is the complicated formation of correct
disulfide bonds by pairing 24 cysteine residues in each TNFrED molecule.
We used the yeast cell surface display method to identify mutants of
TNFrED, which are more abundantly expressed than wild-type TNFrED. Display of
mutant libraries on the yeast cell surface has been used to identify mutants
of both single-chain antibodies and single-chain T-cell receptors that have a
higher affinity for its respective ligands and are also used to improve the
level of expression of a single-chain T-cell receptor
(912).
However, the yeast surface display system has not been used to study
multimeric proteins. We describe the isolation of two novel proline
substitutions that increase the expression level of TNFrED in both yeast and
mammalian cells. These mutants do not have an altered affinity for
TNF-. Analysis of structural roles of the mutants in the study has
revealed an interesting structure-expression relationship.
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EXPERIMENTAL PROCEDURES |
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Expression of TNFrED on the Surface of Yeast CellsSaccharomyces
cerevisiae strain BJ2168 (a, prc1-407,
prb1-1122, pep4-3, leu2, trp1, ura352; Yeast
Genetic Stock Center, Berkeley, CA) was transformed with either pYES2 or
pYES2-TNFrED-Agg using the lithium acetate method previously described
(13). Transformed yeast cells
were grown overnight with shaking in Ura medium supplemented
with 2% glucose at 30 °C. Expression was induced by growing the
transformed yeast overnight at 30 °C with shaking in Ura
medium containing 2% galactose and 1% raffinose. Cells were harvested by
centrifuging at 16,060 x g for 2 min, washed twice with PBS
(Invitrogen), and then diluted to 4 x 106 cells/ml. 4 x
105 cells were incubated with either biotinylated human TNF-
(50 or 10 nM) or goat anti-human sTNF RI antibodies (0.7 µg/ml,
R&D Systems) or both for 1 h at room temperature. Human TNF-
(Protein Purification Group, Serono Reproductive Biology Institute) was
biotinylated with the EZ-link Sulfo-NHS-LC-Biotinylation kit (Pierce). Cells
were then centrifuged at 16,060 x g for 2 min and resuspended
in ice-cold PBS containing 0.1% bovine serum albumin. FITC-labeled avidin (2.2
µg/ml, Jackson ImmunoResearch) or R-phycoerythrin-conjugated donkey
anti-goat IgG (2.2 µg/ml, Jackson ImmunoResearch) or both were added to
cells and incubated at 4 °C for 45 min. Cells were centrifuged at 16,060
x g for 2 min, washed once with ice-cold 1x RDF1 buffer
(R&D Systems) and analyzed on Becton Dickinson FACSort. The event rate was
set at
150 cells/sec, and a total of 10,000 cells were collected per
analysis. The yeast population was gated according to light scatter to avoid
analysis of clumped cells.
Production and Selection of Random Mutant LibrariesFive
unique restriction endonuclease recognition sites were introduced into the
coding region of TNFrED by silent mutagenesis using the GeneEditor in
vitro site-directed mutagenesis system (Promega). This step was completed
in order to divide the TNFrED into 6 regions of between 40 and 105 bp. Five of
six regions were separately subjected to a modification of a random
mutagenesis method previously described
(14). Primers flanking the
mutated, long oligonucleotide were used to amplify the DNA by PCR into
cassettes for each region of TNFrED. Randomly mutagenized DNA regions were
digested with the appropriate restriction endonucleases, ligated into the
pYES2-TNFrED-Agg construct, and transformed into XL10-Gold ultracompetent
cells (Stratagene Corp.). Approximately 5 µg of plasmid DNA from each
random library was transformed into ten aliquots (1 x 109
cells/aliquot) of BJ2168 cells using the lithium acetate transformation
method. Cells were grown for 2430hat30 °C with shaking and then
1 x 108 cells were induced for expression.
For each fluorescence-activated cell sorting (FACS) experiment, 4 x
106 cells were labeled as described above using biotinylated human
TNF- at a final concentration of 50 nM for the mutant
library containing mutant clone 6 or 10 nM for the other mutant
libraries. Goat anti-human sTNF R1 antibodies were added along with
FITC-avidin and R-phycoerythrin-conjugated anti-goat IgG. A total of 1.2
x 107 cells were sorted for each library. FACS was completed
on a Becton Dickinson FACsort at an event rate of <2000 cells/sec. The
first round of sorting was performed in exclusion mode and subsequent sorting
was completed in single cell mode. Selected cells were re-grown at 30 °C
and then induced in galactose/raffinose selection medium for the next round of
sorting. Each library was sorted a total of 34 times. Approximately
0.080.4% of cells were collected in the first round, and
0.010.2% in the subsequent rounds. The collected cells from the last
round of sorting were plated on Ura plates to yield single
colonies.
Recovery and Analysis of Mutant TNFrED ClonesApproximately
50 individual yeast clones from each library sort were analyzed by flow
cytometry. Induced cells were incubated with either 50 or 10 nM
biotinylated TNF- under the same conditions as described above. Binding
of TNF-
was detected with FITC-avidin (2.2 µg/ml), and cells were
analyzed by flow cytometry on the Becton Dickinson FACsort. Clones having a
greater median fluorescence than yeast expressing pYES2hTNFrED-Agg control
were chosen for rescue of plasmid. DNA plasmids were recovered from yeast
(15), and transformed into
competent E. coli JM109 (Promega). Purified plasmid DNA was
re-transformed into BJ2168 yeast cells, and individual clones were re-analyzed
using methods described above. The TNFrED regions of positive clones after the
re-transformation were sequenced using the Thermo Sequenase-radiolabeled
terminator cycle sequencing kit (Amersham Biosciences).
TNF- Binding AssayYeast expressing either
the wild-type or mutant TNFrED-agglutinin fusion or yeast containing the
control vector pYES2 were resuspended in PBS/bovine serum albumin (10 mg/ml)
at a concentration of 1 x 108 cells/ml. In each well of a
Durapore 96-multiwell plate (Millipore Corp.), 50 µl of the cell suspension
were incubated with 50 µl of PBS/bovine serum albumin (10 mg/ml) containing
various concentrations of 125I-TNF-
(Amersham Biosciences)
for 2 h at room temperature. Following the incubation, the wells were washed
three times with ice-cold PBS using the MultiScreen filtration system
(Millipore Corp.). Nonspecific binding was determined at each concentration of
125I-TNF-
with the yeast containing the control vector
pYES2, and the nonspecific binding was <10% of total counts. The
Kd and Bmax was determined
using the GraphPad Prism program.
Expression of TNFrED Mutants in Mammalian CellsThe pCMVhTNFrED constructs were transiently transfected into HEK293-EBNA cells using the calcium phosphate method. Transfections were completed in triplicate, and 48 h after transfection the level of hTNFrED in the conditioned medium was measured with an ELISA for hTNF R-1 (R&D Systems).
Quantitative RNA AnalysisTotal RNA was prepared from transiently transfected cells using the method previously described (16). Reverse transcription reactions were preformed using 2.5 µg of total RNA and the SuperScript First-Strand Synthesis System (Invitrogen) following the manufacturer's instructions. TNFrED mRNA expression levels were determined by real-time PCR analysis using primers 5'-TCCAGTGCTTCAATTGCAGC-3' and 5'-TCTGTTTCTCCTGGCAGGAGA-3', and probe 6FAM-TCTGCCTCAATGGGACCGTGCA-TAMRA in a fluorogenic 5'-nuclease assay (TaqMan, PE Applied Biosystems). 18 S rRNA expression levels were quantified using the predeveloped TaqMan assay for eukaryotic 18 S rRNA. 18 S rRNA levels were used as an internal control, and TNFrED mRNA levels were normalized to 18 S rRNA levels. The average threshold cycle was calculated using the ABI PRISM 7700 sequence detection software.
Surface Plasmon Resonance Analysis of TNFrED
MutantsSurfaces displaying polyclonal goat anti-human TNFrED were
constructed by binding biotinylated antibody (BAF225 from R&D Systems) to
a Sensor SA chip (P/N BR-1000-32, BIAcore Inc.), which was conjugated with
streptavidin. Biotinylated antibodies bind to the chip with high affinity and
create a stable surface for repetitive capture of TNFrED, TNF binding
analysis, and regeneration. TNFrED concentrations of conditioned media
containing the mutant TNFrEDs had been measured by ELISA prior to submission
for binding analyses. Buffer-diluted purified TNFrED or buffer-diluted
conditioned medium containing TNFrED was injected onto the chip surface at 50
nM, resulting in the formation of an antibody-TNFrED complex.
TNF- at 1 nM (trimer) was then injected, and the kinetics of
the binding and dissociation were recorded via the time course of surface
plasmon resonance response. The chip surface was regenerated with 50% 100
mM sodium citrate, pH 2.5, 50% 100 mM sodium citrate, pH
3.1, which stripped off the TNFrED and the TNF-
. This series of
injections was repeated for TNF-
at 2, 5, 10, 20, 50, and 100
nM while holding the concentration of injected TNFrED or mutant at
50 nM. Each set of binding curves was fit using global analysis and
a 1:1 interaction model.
Structure-Expression Relationship AnalysisThe structures
were downloaded from the May 2002 release of the Protein Data Bank (PDB)
(17) at the Research
Collaboratory for Structural Bioinformatics
(www.rcsb.org/pdb/).
The PDB codes of the three crystal structures containing TNFrED molecules are
1ext
[PDB]
(18), 1ncf
[PDB]
(19), and 1tnr
[PDB]
(20). The -
angles
of interesting residues in these structures were calculated and were compared
with the
-
angles of the same residue type in a large set of
representative structures in the PDB. A total of 4930 representative
polypeptide chains were selected based on the Astral list (release 1.59) of
SCOP domains using an identity filter of <50%
(astral.stanford.edu/scopseq-os-1.59.html)
to remove closely related structure folds or very similar sequences
(21). The structures in the
list were then limited to those with resolution 2.4 Å or better and
R factor < 22%, which narrowed them down to 2674 structure
domains. Residues in the selected domains were further subjected to B factor
restrictions. Only those with an average B factor of main-chain atoms
(C
, C, O, and N) between 1.0 and 25.0 are qualified for Ramachandran
plots. Each Ramachandran plot was divided into 10° x 10° grids,
resulting in 36 x 36 pixels. The conformation in a pixel is considered
as accessible if the number of the data points in the pixel exceeds the
expected value, which is defined as the ratio of the total number of data
points to the total number of pixels.
The formula of estimating the approximate entropy gain of protein folding
for a mutation from residue X to residue Z was adopted from Ref.
22. The contribution to the
entropy of folding residue Z from unfolded states to its folded structure
relative to residue X in the mutation of X Z is given by,
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RESULTS |
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Selection of Mutant TNFrED Clones with Enhanced
Biotinylated-TNF- BindingA modification of the
mutagenesis approach previously described
(14) was used to generate
random mutant libraries. In this approach mutant oligonucleotides are produced
by spiking a predetermined level of the "wrong" nucleotides at
each position. The level of contamination of the wrong nucleotides was
adjusted to generate either an average of two or three random point mutations
per oligonucleotide. Each mutant library covered between 40 and 105 base pairs
of TNFrED. Ten random clones from each library were sequenced. The type and
position of mutations were random, and the regions were found to contain the
anticipated average number of mutations. The size of each library was between
0.5 x 106 and 10 x 106 independent mutant
clones. Each library was transformed into the strain BJ2168, and
1
x 106 independent transformants were selected for binding to
both biotinylated-TNF-
and polyclonal antibodies directed against the
TNFrED. The window of the two-dimensional fluorescence histogram that was used
to select for the subpopulation of yeast expressing active TNFrED is shown in
Fig. 1C. This window
was originally chosen to select for mutants with an increased affinity for
TNF-
. The selected subpopulation of yeast was grown and reselected with
two-color sorting. After several rounds of two-color sorting the population of
yeast in the selected window was enriched
(Fig. 1C). Following
three to four rounds of cell sorting, individual clones were analyzed by
examining binding of biotinylated-TNF-
to the TNFrED on the cell
surface. A majority of the clones analyzed from each sorted library appeared
to bind higher levels of biotinylated-TNF-
. The mutant TNFrED plasmid
was recovered from each yeast clone and re-transformed into the BJ2168 strain
and then reanalyzed by flow cytometry. The vast majority of yeast clones were
false positives as the recovered plasmids did not confer higher levels of
biotinylated-TNF-
binding. When false positive yeast clones were
analyzed in the absence of biotinylated TNF-
and avidin-FITC, they were
shifted as compared with the parental BJ2168 strain. These false positive
yeast clones are
30% larger compared with the parental BJ2168, which is
presumably what gives rise to the shift in the baseline absorbance. However
two mutant clones, 6 and 11, from different libraries, were identified after
re-transformed into the BJ2168 strain as conferring higher levels of
biotinylated TNF-
binding and therefore are true positives
(Fig. 1D).
Characterization of Mutant Clones 6 and 11The TNFrED coding
region in mutant clones 6 and 11 was sequenced and, as anticipated, the
mutations for each clone were only found in the sequence region mutated for
that specific library. In mutant clone 11 there was a point mutation that
resulted in Ser Pro change at position 87, and in mutant clone 6 there
were two point mutations that resulted in an His
Pro change at position
34 and a Ser
Ile change at position 57. To determine whether the
mutations increase binding of biotinylated TNF-
through increased
expression of TNFrED or by increasing the affinity for TNF-
, we
performed a saturation binding experiment on yeast expressing either mutant
clone 6 or 11. Analysis of saturation binding experiments
(Fig. 2) indicated that yeast
expressing either mutant clone 6 or 11 express higher levels of functional
TNFrED than wild-type TNFrED and that the presence of these mutations does not
affect the affinity of TNFrED for TNF-
. The number of receptors/cell
for yeast expressing mutant clone 6, mutant clone 11, and wild-type TNFrED was
3930, 1490, 740, respectively.
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Expression of Mutated TNFrEDs in Mammalian CellsWe next determined whether the mutant clones derived in yeast had similar expression characteristics in mammalian cells. The TNFrED mutants were transiently expressed in HEK293-EBNA cells and the amount of secreted TNFrED was measured with an ELISA specific for TNFrED. The results (Table I) indicated that either mutation H34P or S87P increased the expression level of TNFrED. Moreover, the relative increase in expression is similar to what was seen when these mutants were expressed in yeast (Fig. 2). The S57I mutation alone did not alter the expression level of TNFrED (Table I). The effects of these mutations do not appear to be additive because the presence of both H34P and S87P on the same construct did not increase the level of TNFrED secreted in comparison to that found with H34P alone.
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Steady-state RNA levels from transiently transfected HEK293-EBNA cells were analyzed by real-time PCR. The RNA levels of TNFrED for the six mutants were found to be 2873% of the level of wild-type TNFrED RNA indicating that the presence of the described mutations does not increase RNA levels.
The association and dissociation kinetics of TNF- binding to the
mutated TNFrEDs were directly comparable to those of the wild-type protein
(Table II). The difference in
TNF-
affinity between wild-type and mutated TNFrEDs was not found to be
significant, which is consistent with our binding analysis with the yeast
transformants.
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Analysis of Accessible Conformations of the MutationsThe residue substitutions in mutant clone 6 and 11 and their adjacent residues include proline, cysteine, and glycine. As these three residues are known to have a significant role in protein structure, we analyzed potential functions of the substitutions or the adjacent residues in their protein-folding processes. We first compared the accessible conformations of the substituted residue types before and after the mutations as seen in the PDB structures. As shown in Fig. 3, the conformation of a proline residue is more restricted than the other three residues, and a serine residue is less restricted than an isoleucine residue. Moreover, the conformation of Ser87 and His34 in TNFrED crystal structures (red-filled marks) lies in the most abundantly accessible conformational region of the proline residues. Following the same method as described by Matthews et al. (22), we estimated the contribution of the entropy of protein folding before and after the mutations: 6.1 J/mol·degree for S87P, 7.1 J/mol·degree for H34P, and 1.66 J/mol·degree for S57I. In all three cases, each mutated residue contributes favorably to the entropic contribution to protein folding (Fig. 3).
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We next analyzed the accessible conformation space of the adjacent residues
affected by the mutations. Conformational restriction has little effect for a
residue following proline (compare Fig. 4,
A and B). In contrast, conformational
restriction of a residue preceding proline is particularly profound due to the
steric hindrance, as illustrated in Fig. 4,
C and D. The rigidity of the pyrrolidine ring of
the proline residue constrains the angle of its preceding non-glycine
residue to a positive value in the top left quarter of its Ramachandran plot
(refer to Fig. 5B).
Glycine is another residue with a noticeable shift of accessible
conformational space when it is adjacent to bulky residues. When it follows
the bulky asymmetric residue, isoleucine, the accessibility of glycine to
conformational space of the small
angle region is notably reduced
(compare Fig. 4, E and
F and refer to Fig.
5C).
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To assess the effect of adjacent residues in the choice of conformation
preferences by the substituted residue, we analyzed the accessible
conformational space of the same neighboring residues in the PDB structures as
those in clone 6, clone 11, and the wild type. The sequences surrounding the
mutated residues are Ser86-Pro87-Cys88,
Cys33-Pro34-Lys35, and
Glu56-Ile57-Gly58, where the
superscript numbers denote residue numbers in the protein. The corresponding
sequences in wild-type TNFrED are
Ser86-Ser87-Cys88,
Cys33-His34-Lys35, and
Glu56-Ser57-Gly58.
Fig. 5 compares the Ramachadran
plots of neighboring residues before and after the mutations. The mutation
from Ser to Pro at residue 87 markedly limited the accessible conformations of
the preceding serine residue to the neighborhood of the observed serine
conformation in the TNFrED crystal structures. Similarly, the mutation from
His to Pro at residue 34 also restricts the accessible conformational space of
the preceding cysteine residue to those surrounding the folded conformation.
This indicates that nearly all of the possible conformations of this cysteine
residue in unfolded states are similar to the folded conformation. In
contrast, the mutation from Ser to Ile at residue 57 reduces accessibility to
small angle conformations of the following Gly residue, although the
small
angle is its folded conformation.
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DISCUSSION |
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In each mutant clone, we showed that the residue responsible for the higher protein expression levels is a proline substitution next to a cysteine involved in a disulfide bond. Structural examination of the TNFrED structures revealed that these residues apparently are not involved in ligand binding, glycosylation, or structural roles of intra- or intermolecular interactions by side-chains. Using an approach similar to that described by Matthews et al. (22) who analyzed the contribution of entropy change to protein folding before and after a mutation, we demonstrated that the entropy change of the substituted residues in the mutations S87P, H34P, and S57I contributes favorably to the protein folding of the mutants as compared with the wild type. The degree of contributions is: H34P > S87P >> S57I.
In addition, we showed that the accessible conformational space of the
residues adjacent to the mutated ones contribute to the protein folding either
favorably or unfavorably. In the mutant S87P, the accessible conformational
space of the preceding Ser residue is limited, and this limitation favors the
protein folding of the mutant relative to the wild type. The higher expression
level of S87P is most likely the result of the positive entropy contribution
of the mutated residue per se and the favorable accessible
conformations of the preceding Ser residue. In the mutant H34P, the Cys-Pro
combination restricts the conformation of the preceding Cys residue to or near
the folded conformation. The correct orientation of the cysteine presumably
facilitates proper disulfide bond formation and results in the higher yield of
correctly folded molecules that lead to the higher expression. In the mutant
S57I, the bulky Ile residue reduces the number of small angle
conformations accessible to the following Gly residue, even though the small
conformation is the folded structure. This negative contribution from
the following Gly residue eliminates the positive contribution of the entropy
change from the mutated residue in protein folding of the mutant. This is
consistent with the observed expression level of S57I, which is actually
slightly lower than the wild type. We also noticed that the expression level
increase is non-additive when both H34P and S87P are present on the same
construct, possibly because the two cysteine residues are not disulfide
bridged to each other.
In summary, this study provides a step forward toward the solution of expressing a potentially important therapeutic molecule, TNFrED. It is also the first application, to our knowledge, of the yeast display system to study multimeric proteins. Moreover, this work presents the first analysis of the structure-expression relationship by demonstrating protein conformation is a determinant of expression.
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FOOTNOTES |
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To whom correspondence may be addressed: Serono Reproductive Biology
Institute, One Technology Place, Rockland, MA 02370. Tel.: 781-681-2705; Fax:
781-681-2942; E-mail:
xuliang.jiang{at}serono.com.
To whom correspondence may be addressed: Genzyme Corporation, One Mountain
Rd., PO Box 9322, Framingham, MA 01701-9322. Tel.: 508-271-2847; Fax:
508-872-9080; E-mail:
william.brondyk{at}genzyme.com.
1 The abbreviations used are: TNF-, tumor necrosis factor-
;
FACS, fluorescence-activated cell sorting; TNFrED, extracellular domain of the
p55 TNF receptor: PDB, Protein Data Bank; PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate; ELISA, enzyme-linked immunosorbent
assay.
2 W. H. Brondyk and M. Cunningham, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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