Note: Descriptions are shown in the official language in which they were submitted.
17/~)6'~ 53 ~171 242 ~g~ ABEL ~ IMRAY 2 0 4 4 717 ~0~4
PROC~SS ~oR--THE-~EpARATIoN OF
PRo~EINS, POL~PEPTIDES OR METALS
The pre~ent invention relate~ to sepa~ation of
polypeptides or proteins or re~oval of metal ions, and to
chromatographic agents ~uitable therefor.
Chro~atographic agent~ fo~ u~e in variou~ separation
proces~Q~ ~r~ w~ll kno~n ~n the ar~. However, there has
been a treme"dous need for chromatoara~hic ~ents w~th
specific affinlty for proteins. A fe~ proteins such a~
lectins and protein A are u~e~ a~ ligand~ for af~inity
eeparat1o~. Howa~cr, their u~e 1~ limlted: lectins c~n
be u~ed only for the isolation and purificatLon of
gly~oprot~in~, and protein ~ only for thç i30l~t1on ~nd
purif iaation of immunoglobulins.
The applicants have discovered that immobilised
rhr~c~ * m~lil~d phoc~'~'~ ~ol: a~ o~oollort:
chromatograph~c agent~ for the sepa~a~ion of polypeptides
and protein~, especi~lly those that have a high affinity
L~ uti~ u~ rln~ ~luster6.
It is known that pho~vitin ha~ a high affinity for
metal ions, The applic nts have found al~o that thi~
property is al~o exhi.~ited by immobilised phosvitin,
which c~n be ~tilis~d in a ~y6tem w~rking on the
principl~e of mct~l-chcl~te ~hromato~r~ph~. M~tnl ion6
25 su~h, for example, a~ Fe3+, Fe2~, Ca24 Mg2+ Mn2+ Cu2+
Co2+ and Zn2~ are all biolo~ioally very ~mportant becau~e
17,'~! 9~ 54 ~:~71 242 ~Y~ ABEL ~ I~IRAY 2 0 ~ 4 7 17 ~1~05
of thQir involvement in a variety of catalytiC processe8.
Accordingly, the pre~ent invention provide5 the use
of phosvitin or a modif ied Phosvitin for the ~reParation
of a chromatographlc agent for tho ~ep~ration ~nd/or
purification of polypeptide~ and protein~ or for the
removal of metal ions from biolo~ical material.
The preE;er~t invention also pro~i~lee phosvi~in or a
modified pho~vitin immob~ ed and coupled to a ~uitable
matrix, for use in the separation and/or purification Of
polypept~des and proteins.
The phosvitin or modif ied pho~vitin may ~ave been
produced by recombinant DNA technology, and the ter~
"pho~vitin~ as used herein includes both mol~cules of
natural origin and the corre~ponding recombinant
molecule~. In the case of a modifled pho~vitin, t~e
modifled molecule itself may be produced by recom~inant
DNA technology, or recombinant phosvi~in may be produced
and then ~odified.
A modi~ied p~o~vitin may have, for example, one or
more of the following modifica~io~s while still preserv-
ing binding ability: removal of 60me or all of tho
carbohydrate, removal of one or more amino acids;
addi~ion of one or more amino acids; modif ication at one
or more individual amino acid re~idues, replacement of
one or more individual amino acid reeidu~6, ~or example
of a~partic acid by gluta~ic acid ~r lysine by arginine;
appropr~ate phy~ical change to the molecule.
17 ~ ~1 19 3~ ~)71 2~2 Yg~9 ABEL & IMRA~ 2 0 4 4 717 ~ )2
If decired~ the phosvitin or modified phosv~tin may
be in the form of a metal chelate complex.
The phosvitin or modi~ied phosv~tin may be used in
the i~olation of polypeptides and proteins, for the
separation of various individual polypeptides and
proteins from their impurit1es, and for the p~r~f$cation
of polypeptides and protein~. Thus, for exa~ple, the
chromatographic agent may be used for the resolution of a
mixture o~ protein6 ~r polypeptides from a broth or an
lo extract, and/or it may be used to obtain a protein or
polypeptide in a substa~tially pure ~homogeneou~ form.
A non-chelated phosvitin or modi~ied pho6v~tin may
also be used for the rem~val of metal ion~ from biolo~i-
c~l material, e~pecially from physiological ~luids, for
example from blood, serum or pl~sma.
The present invention further provides a process for
the 6eparation and/or purification of a polypeptide or a
protein, or ~or the removal o metal ion~ from biological
material, wherein there is used as chromato~raphic agent
pho~vitin or a modified phosvitin, immobili6ed and
coupled to a suitable matrix.
PhOsvltin is rich in pho~phorylated ~erine resldu~
and the~e normally occur in clusters in that prDtein.
~e appli~ant6 have found by exa~ination of X-ray
orystallography data, in relation to protelns havinq
eerihe clu6ters, t~at the conf iguration o~ tho~e clusters
varie~ ~reatly from prote~n to protein, implyinq, they
17/01; 91 Ig 35 S071 2~12 Y8.'~Y IRA'I 2 0 ~ 4 71 7
believe, that the amino acid~ in proximity to the
the Configuration tho
can assume.
have al~o diSC~Vered th t
XamPle, Cytochrome-c ly~
human FOlliCle Stimulating hormon~ S~I~ hav8 ~.n
inity ~or the phospho
~5 immohilised ~orm Th
~u~h as Cytochrome-C and ly~o2yme carry charge ~ ter~
near their C-terminal region8~ and the applic~nt6 ~elieve
u~ter regions ~hich com 1
pho~pho-8erine cluster domains on pho6vitin are inv~ed
in the ob~erved affinity phenomenon for these proteins.
It is considered that the binding effect~ ob~e~ved
re8ult of a generalised
he Po~yanionic phO8viti
and a polycationic protein or polypeptlde. It i3
believed that it i~ the p~rticular structure and co~-
he Phosvitin mlecule t
p erine c:lus'cer2~ that 1
ertain particular prot i
tide~ that have a compleme~tary s~ructUre and ~onfigura-
n the region of charge
A~cordingly, al~o, we believe, ~uitable modified
SPeCiallY tho~ie retaini
rine re3idueg, e~;peciall
rably the majOrity of th
17~ 7 ~7~ y~i') A~EL.~ A~ 2 0 ~ ~ 7 1 7
- 5 -
phosphorylated ~erine residue~ are ret~ined.
Advan~ageou~ly, 5ub~tantially dll such cluRters are
retained.
Further evidence for the pos~ulated involvement of
domains in the interaction i~ given ~y experiments in
which modi~ications of ar~inine residue~ in lysozyme with
diacetyl a~d of lysine residue~ in cytochro~e-C with
acetic anhydride were carried out. ~he applicant~ have
found that the6e modificatio~s lead to comple~e 1068 of
the binding property of the~e protein~ to a phosvi~in-
SepharO~e matrix.
Accordingly, polypeptides and proteins that may be
æeparated and~or purified by the non-chelat~d chromato-
~raphic ~gents accordi~g ~o ~he invention are e~pecially
tho~e having charge clusters, includin~, for example,
various growth factor6, D~A blnd~ng proteins (~hose
involved in early gene repliaation a~d transcrlption
proce~es3 and DNA- and RNA-modifyin~ enzyme~. Examples
include those ~iven in the following Ta~le 1:
T~
A. ~rowth ~ormone~/~aCtors:
a) AdrenocortiCOtrOpiC hormone
~) Parathyroid hormone
c) Fibrobla6t gro~th factor~ (both acidic & baQic)
d) Astroglial growth factors 1 & 2
e) Retina-deriv~d gro~th factor
f) Eye-derived growth factor
I7~I IS~5~ ~071 ?4Z Q`3~Y ABEL & IMRAY 2044717
- 6 - !
g) ~artilage-derived growth factor
h) End~thelial cell growth factor
B. DNA b~nding protein~:
n) Pro~ n6 having POU do~ain~
b) Proteins having Romeo domains
c) Zi~c-finger proteins
d) Leucine Zipper protein~
e) Amphipathic helix-loop-helix motif-containing
proteins
C. PNA-modifying enzymes
D. RNA-modifying enzymeo
E. DNA-recombinant fusion protein products:
Since certain domains in pro~eins ~uch ~
cytochrome-C and lysozyme have strong affinity for
phosvitin, it ~s considered that t~e engineering of
the genes corresponding to tho6e doma1n3 along with
the gene coding for a protein of interest into an
organi~m to produce ~ fu6ion proteln containing
tho~e do~ains will facilitate their rapid and
specific purification using the phosvitin/modified
phosvitin chromatographic agent according ~o ~he
invention .
It has been p~oposed to use immobilised phosvitin to
purify protein kina~e~, for which enzymes phosv~t~n can
act as a substrate. Such u~e i6, however, limited to
that p~rtic~lar class of enzyme6, and is not in~luded in
the pre~ent in~ention.
~ 5~ ~n71 ~ 9S~ A~EL ~ IMRAY 2 0 4 4 717 ~ 3
The chemical and st~uctural feature6 o~ phosvitin in
unmodified o~ modified form that make it eminently
~uitable for use as chromatoqraphi~ ligAnd based, it is
believed, on the principle of charge clu~ter interaction~
also lend them~elves ~ the qeneration of metal-chelates.
This ability to form metal chelates is of use, not
only for removal of metals from biological and non-
biological ma~erials, but al60 for qeneration of a metal-
chelate ch~omatographic medium, and this may be u6ed in
the ~eparation and/or purification of both biological an~
non-biological material~ Thu~, we believe that metal-
containing pro~eins and tho~e having high affinity for
metal ions may be purified by metal chelate
chromatography.
Thu6, for example, the appllcant6 h~ve foun~ that a
pho6vitin-sepharo~e matrix wit~ ~ppropriate chelation
with zinc result~ in a complex which exhiblt~ affinity to
proteins: for example it ex~ibited ~trong blnding of at
lea~t two proteins from egg white (110 Kd and 120 Kd).
Similarly, chela~e complexes with ~3+, F~2+, CU2+ nnd
Ca2+ bound to pho~itin may be prepared and used for the
purificat1On of ~etal-dependant enzyme~ and oth~r
protein~, an~ an iron chelate complex of pho~vitin
affinity ma~erial may be u~ed to remove peroxide3 from
~olven~s,
The high affi~ity exhibited by pho~vitin in
unmodified or modified form toward~ ~etAl ions indica~e~
l) ~071 ~4~ ~`3~ ABEL ~ IM~lY 2 0 4 ~ 7 1 7
that the p~osvitin matrix may al~o be u~ed for ~cavenging
exce6s metal ~ons from physiological fluidA, for example
by haemodialysis. ~or example, in iron-overloa~ states,
the phosvitin matrix may be used, for example for ~ron
scavenging from serum in Cooley'8 anaemia.
Phosvitin is a naturally-ocGurring protein, found
in avian and fish eggs. Phoevitln can be obtained in
purified form (electrophoretically homogeneous) by a
n~mber of techniques known eeE ~e, ~or example a~
described ~n J. Am. Chem. Soc. tl949] 71, 3670. Thus a
chromstographic agent used in the present invention has
the advantage that it ~tilises a protein which i8
naturally abundant and whi~h can be purified with
relative ea6e.
lS Adv~ntageously, the ~-form o~ phoBvitin may be u~ed;
this has a higher phosphate con~ent than the ~-form.
~ he invention lnclu~s al~ U~Q o~ mod1~led
forms of pho~vitin, whether or not prepared from phos-
vitin itself, and as well ae the possi~ility of using a
modified pho~vitin a~ inltio, the po~sibility of carrying
out one or more modification~ at any suitable sta~e in
the preparation of the chromatographic agent may be
mentioned.
Modified p~losvi~ins hav~ng affini~y for metal ions,
for example substantially the same affinity ac has
pho~vitin, ehould especially be mentioned.
Modification to chain length may be carried out, for
20~717
17~6'gl 19`~1 ~Q71 242 ~9S~ AB~L ~ IMRAY ~12
_ g _
example, by chemical and/or enz~ma~ic mean~, for example
proteolysi~ with the protea~e trypsin. It is surprising
that thi6 proteolysis ha~ proved po~s~hle, becau~e i~ i~
generally con~idered that phosvitin ig r~sistant to
proteoly6is. Phosvitin modified in thi6 way and
immobili6ed on a ~uitable matr~x can have an e~pecially
high binding capacity.
Other chemical andlor ~nzymatic modification6
directed at specific amino a~d re6idues ~re also
possible. Furthermore, if t~e gene for pho6Vitin is
cloned into another organism ~o produce a recombinant
phosviti", ~ite-directed-mutagene~i6 may be used to
~hange a particular amino ~c~d, for example a~partic acid
to qlut~mic acid or lyRine to ar~lnine~ Such modi~ica-
tions are well-known in molecular blology. If a result-
ing re~ombinant phosvitin or modified pho6vitin molecule
i6 not already phosphorylated, a phosphorylation reaction
~hould generally be carried out.
The phosvitin or pho6vitin modified for ex~mple as
above may be in the native glycosylated form, or it may
be partly or ~ully deglyco~ylated. DeglycQsylatiOn
methods are described in the literature. For removal of
asparagine-linked (N-linked) ~lycosylated m~etie~ see
Tarentino A.L., ~omez ~.M. and Plummer T.H., (1985),
Biochemi6try, 24, 4665-4~71; for removal of serine-and
thrçonine-linked (o-linke~) glyco6ylated moieties 5ee
A~.S.B. Edge, et ~1, (198~), Annal Bio~hem, 118, 131-137.
17~ ' gl 1~ 2 ~l~71 '~'4~ ABEL .~ IM~A~ 2 0 ~ ~ 717 ~013
-- 10 --
Appropriate physical modification Or the pho~;vitin
should al60 be mentioned.
Iwo or more mod~fications m~y be carr~ed out in any
suitable order; fox example, change of amino acid
sequenae and/or of chain length may b~ c~rried out before
or after deglycosylation. U~le~s the context indica~es
otherwise, references herein to "phosvitin" are u~ed to
include modif ied pho~vitin.
The chromatographic agent used according to the
lO invention may be prepared by methods known ~ 6e: see,
for example "A new method ~or the analysi~ of blood 6erum
glycoprote~n~ using Sepharose coupled Lect~ns", S.
Thompson and G . A . Turner, in Lectins, edited by T. C.
B0g-Hansen and D.L.J. ~reed, pp 453-458, publi~hed by
Sigma Chemical Company, 1988. The phosvitin may be
attached directly t~ the matrix or l~directly, by u~e of
cpacer arms.
Thus, a chromatographic agent comprising phosvitin
or a m~dified pho~vitin immobilized and coupled to a
suitable ~atrix may be prepared by a proces3 comprising
m~xing the phosvitin wi~h the matrix in the presence of a
buf f er so that ~he pH of the mixtute is in the range
prefera~ly of from 8.0 to ~.3, washing awHy the exce6s
ligand of the phogvitin and then ~locking the ~emaining
2S active gr~ups of the matrix by treating the mixture with
an amine to produce coupled p~osvit1n-matrix, washing the
re~ulti~g produc~ and recovering the coupled phosvitin-
!
I
17~0t~ ' Yl 1~)3 ~71 ~4;> 9~ 3 ABEL ~ IMR~Y 2 0 ~ ~ 717 ~1~14
-- 11 --
matrix.
The matrix may be, for example, a sepharo~e gel. Wehave used CNBr-activated Sepharo~e and fou~d that it
efficiently couple~ and immobllise~ pho~vitin and
modified pho~vitin. Activation of Sepharose with
cyanogen bromlde and coupling of protein~ ~uch as le~tin~
to such activated matrix is wel~ known in the a~t. In
a~dition to cyanogen bromide-activated-Sepharose, othe~
support media/matrice6, ~uch, for example, as a~arose,
acrylamide, silica and suitabl~ fibres (both syn~het1c
and natural) which are appropriately mo~ified to enable
coupling of proteins, may be used. The use of spacer
armg usually employed for coupling con~aining 6 to 12
carbon atoms, for example ~-amlnohexanoic acid or 1,4-
lS bis ( 2, 3-epoxypropxy) butane, to increase the binding
capacitie~ of the affinity material may provide an
add it i ona 1 advantage,
The weight ratio of phosvitin to matrlx u~ed may be,
for example, substantially 0.005:1. The u~e of 6 to
10 mg of phosvltin p~r ml of ~wollen activated Sepharose
or other matri~ is recommended. The maximum amo~nt o
coupling we have observed i. about 6mg/ml; this range of
protein gives an affinity product of suff~clen~ capacity
for example for puri~ication of proteins.
Mixing of the phos~itin and matrix may ~e carried
out, for exampls, at a temperature in the range of fro~
4 to 25~C, more e-~pecially at ~ubstantially 4~C.
17~ 71 2~2 ~ ABEL .e I~IR,~ 2 ~ ~ ~ 717 ~1~l5
The coupling of the phosvi~in to the matrix is
carried o~t in the presence of a suitable buffer. ~or
acti~ated matrices reactive to amine function6, this
buffer ~hould preferably be free of primary amines, since
if the coupling buffer contain~ reactive amino groups
along with the protein lig~nd to be coupled, then the
extent of protein ligand attach~ent to the matr~x will be
rodu~, r~ult.in~ in lower ~finl.ty cDpncity~ Any
buf rer lacking an amino ~roup may be employed, although
most preferred are 60dium bicarbonate and borate buffers.
In gener~l the buffer should provide a pH in the range of
from 8.0 to 8.3. Excellent re6ult6 may be ohtained
employing ~od~um bicarbonate buf~er containing about 0.5
M NaCl. The use of a 0.1 M NaHC03 buffer having a pH of
substantially 8.3 and containing 0.5 ~ NaCl should
especlally be mentioned.
A~ter coupling with the matrix, exces6 ligands are
washed away, for example with the buff~r used for the
coupling, and as soon as prac~icable thereafter the
remaining active groups are blocked, generally with an
amine or an amino acld. Primary amines are preferred.
Especially good re~ults have been obtained using ethanol-
amine. Amino acid~ may also be used to block exce5s
reactive sites but are less preferred as these would
introduce unwante~ charge~. The amine may be used, for
example, in a con~entration of from o.l t~ 1.0 M; 1 M
ethanolAmine is especially suit~ble,
17~J6 `gl 19:0~ ~)71 242 ~9~ ABEL ~ 1MRA~ 204~717
The blocking ~eaction may be carried Oue, fo~
example, at a pH of from 7.5 to 9.5, more especially at
a pH of sub~tanti~lly 9. It may be carried out, for
example, for a period of from 2 to 18 hour~, for example
for substantially 16 hourQ. Suitably a temperature in
the range of from 4 to 25c, for example 6ub6tantially
40C, is used. Blocking with l M ethanol~mine at a pH o~
9 for 16 hours at a temperature o~ 4C should ~pe~ially
be mentioned.
~he resulting co~pled pho~vitin-matrix i8 then
generally ~a6hed, to remo~e non-specifically bo~nd
proteins, if any, from the matrix. usually, two diff-
erent pR~ are u#ed, more e~pecially two or mor~ cycles of
alternatlng pH. The number o~ c~vcle~ 1q g~A~rally three,
alt~ough the ~umber of such cycles is not critlcal.
Three wa~hing cycles, for example, may be c~rried out,
each cycle oonsisting, for example, of O.l M acetate
buffer p~ 4.0 containing 0.5 M Na~l, followed by a wash
with O.l M Tris-HCl pH 8.0 containing 0.5 M NaCl. The
applicants have found that three su~h wa~hes generally
remove non-~pecifically bound proteins (i~ any), from the
matrix, a~ determined by absorbance at 280nm. The
chromatographlc agent ~ay then be recovered b~ fil~ra-
tion. Washing and reco~ery procedure~ used in coupllng
protein6 to act~vated matrices are well known.
Preparation of a chromatography column may ~hen be
carried ou~ by known methods. ~or example, af~er
17~ 1 19~ 171 24~ Y A~EL ~ IM~AY 20~4717 ~017
- ~4 -
pac~ing a ~itable column wit~ ~he phosvitin matrix,
equilibration m~y be carri~d out for exa~ple with ~ri~
}~Cl buffer, ~uitably with 10 to 50 mM Tr~ HCl, pH 7.5 to
8.5; a suitable flow rate i6 0.25 mltmin.
To prepare a metal chelate, the column after
equilibration may be txeated with a sui~able buffer
containing metal ion~; ususlly thQ same buffer used for
equil~br~tion iB u~ed ~n this 3tep, fo~ example lOmM ~ris
buffer, pll 7 . 5, con~aining, for example, o. l M zinc
acetate, 0.l M ferric chloride or o. l M calcium chloride.
~wo to three column ~olume6 o~ such ~etal salt-contain-
ing buffer i6 suitably passed through the column, and the
column then equilibrated with buffer alone.
Preparation of beads ~arrying a metal ~helate, for
example an iron chelate, ~or example for peroxide remo~al
from solvent~, ~hould al~o be mentioned. Methods for
the produ~tion of such beads are de.~cribed ih the
literature.
The present invention al~o provides a chromato-
Z0 graphic agent ~hich ~ompr~ses phosvitin in the form of a~etal chelate complex, immobilised and coupled to a
suitable matrix, and a chromatographic agent which
compriseEi a modified phosvitin, if dQalr~d ~n tho ~rm Or
a ~etal chelate complex, immobllised and co~pled to a
suitable matrix, and a process for their preparation as
described a~ove.
Thn ~c~ual process ~or the separation or
7/~)6' g1 1~ ~l7 ~)71 ~42 ~9~9 ABEL & IMRAY 2 ~ ~ ~ 717 Q1~18
-- lS --
purification of proteins/polypeptides or for the r~moval
of metal ions may be carried out ~ccording to methods
known per se, Using ~et protoc~ls, for example as
follo~s.
A column is prepared and equili~rated as de~cribed
above. The column is the~ loaded with the crude material
(containing for example l.0 to 5.0 m~/ml protein or th~
metal-containing materi~l), centrifuged for example at
lO,000 rpm for a period of, for example, 10 to 30
minute~, ~uitably at 4~C. The column i~ then washed with
the equilibration buf~er until all non-binding protein~
are washed, and the column is eluted, for exa~ple with
Tris HCl buffer, suitably with 10 to 50 mM Tris HCl, p~
7 . 5 to 8 . 5, with a linear gradient of O.lM to 2M NaCl. A
fraction o~ suitable volume i6 colle~ed, the wash and
eluants being monitored with absorbance at 280nm. For a
metal chelate compl~x, the pH of the buffer is generally
lowered to obtain elution. For scavenging ~etal~, there
i~ generally no elu~ion by salt, although if proteins are
bound alon~ with salt elution ~y be neces8ary.
Acoordingly, the pre~ent invention espeoially
provides a proces~ for the sepa~ation and/or puriflcation
of a polypeptide or protein, especially one having af~i
nity for phospho-serine, which comprises loading the poly-
peptide or protein onto a chromatogrsphic column contain-
ing a chromatograp~ic ~geht comprising phosv~tin or a
modified pho-qvitin, immobilised and co~pled to a sui~able
17~ l l8~)~ ~071 ~2 ~g~ ABEI.~ I~IRAY
20~717
matrix, previou61y equilibrated with an equ~libratlng
agent, and eluting the column with a ~alt solu~ion to
obtain the polypept~de o~ protein.
For example, for purification of ly60zyme derived
5 from egg white, the equilibrating agent may be 66 mM
KH2P04 at a p~ of ~.~4, and the bu~er employed for the
elution may be 100 mM KH2P04 ~t a pH of ~. 24 containihg
200 mM NaCl.
For purification o~ a re~tr~ction enzyme, the
e~uilibrating agent may be, for example, 10 mM ~rris-~cl
having a pH of 7.5 and containing 50 mM NaCl, 5 mM MgS04
and 1 mM D~T~ and the salt solu~ion may be, for example
10 ~M Tri6-HCl having a pH of 7~5 and Containing ~.5 M
NaCl, 10 mM XCl, 100 ~g/~l BSA and 1 ~M DTT.
The colu~n operation~ ar~ ~ultably performed at 4C.
In the case of enzymes, activitie6 are determined and fo~
hormones recommended immunoassay~ are performed.
The followi~g Examples illust~ate the invention.
Example ~:
~0 Purifi~ation of phosvitin
~ en egg yolk3 were separated and the yolk material
was obtained by puncturîng the vitelline membrane and
draining oU~ the contents. The yolk Content~ (100 ~ms)
were ~u~pended in ~.11 M MgS04, 5.5 time6 the volume of
25 the yolk materi~l, and mixed vlgoro~sly. The m~xture was
l7~(~6 ~1 Ig 0~ ~)71 242 ~9~9 ABEL & I~RAY 2 0 4 4 717 ~02~!
- l? -
kept at 4C for 18 hours to allow precipitation. The
precipitate was di~perSed ~n 70 ml of 0.4 M (NH4)2~04 and
the pH wa~ adjusted to 4Ø The diapersion was mixed
thoroughly and centrifuged. The supernatant was extrac-
5 ted with 30 ml of ether and thi6 wa6 repeated threetimes. After every extraction the aqueou6 layer was
separated. All aqueous fractions were pooled together
and treated with (NH4)2S04 to giv~ 95% ~aturation. The
saturated mixture was allowed to stand at 40C overnight.
The precipitated protein was cen~rifuged and
dissolved 1n water and dialysed against distilled water
fo~ 48 hours at 4 C. The water wa~i changed every 8
hours. The dialysed protein solution wag lyophilized.
Yield s
From lOo gm~ yolk material 290 mg dry protein were
obtained.
The puri~ied phosvitin WB6 then filtered by gel
filtration to separate the ~ and ~ forms . For thi6 a
Sephadex G200 column (104 x 2.5 cm) was equilibrated with
100 mM ~odium acetate bu~fer pH 5 . O and the dialysed
protein (50 mg) wer~ loaded onto the column. ~he protein
in the eluate was monitored ~y OD280. From the analysi6
of the phosphate content of the protein the aecond peak
corre~ponded to the ~ form. The peak ractions were
pooled an~ dialysed against distilled water. ~he
dialy~ed protein s~lution wa~ lyophilized. of the So mg
protein loaded 34 mqs were recovered in the peak
17~ '91 29 1(~ ~)71 '?42 9138~ A~EL ~ IMRAY 2 0 4 ~ 7 ~ 7 ~J21
- 18 -
fractions.
Analysis of phosvitin.
1 Pho~phate (inorganic): Phosphate estimation for
the protein WaA don~ uning ~ilO Amm~niU~ Muly~B~e
Method (Anal. Chem. [1956] ~8, 1756). Tho di~uot
protein (H2So4 + HN03 dige~tion~ w~s 6~bjected to
analy~is. The ~ rorm had 10.7 - 11. 8% pho~phorus.
2 Protein estimation: Lowry's method (J. Biol. Chem.
~1951], 193~ 2~) W~S ~sed for the proteln
estimation.
Electrophoretic analysis~
Electrophoresis was done on 10% homogeneoU~ poly-
acrylamide gel with Tris-~lycine buffer p~ 8.3. ~e
purified protein showed a single ma~or band. Sub~e~uent
anal~6is on S~S-PAGE showed that the molecular welght of
the purified protein was in the range of 3Q,000 to
35,000. ~here were no major impurities as~ociated with
thi~ protein.
Exa~ple 2:
Prepar~ti~o~ ~ chromatoqraphia agent; immQbilisa~i~n and
couplin ~_~ho~ltin ',
3 g of cyanogen bromiæe-activated SephaX~se in
freeze-dried powder form were allowed to swell in lmM HCl
and washed with 1 m~ HCl in a ~intered glass ~or 15 min.
The gel was then wa~hed with dlstilled water. Phosvitin
(15 mg) in the native glycosylated form prepared in
l7~0~ 1l ~l)7l ~4~ ABEL & IMRAY 2~4~717 ~Q22
-- 19 --
Example l was di~olved in 25 ml of O.lM Na~C03 pH 8.3
oontainlng 0.5 ~ NaCl. ~hi~ w~s mixed with the gel tlO
ml) and allo~ed ~o rotate gently overnight a~ ~C. Th~
exce~s ligand ~as wa6hed a~ay with 0.1 M NaHC03 pli 8.3
S containir~g 0.5 M NaCl and the remaininy active groups
were blocke~ by treatment with 1 M ethanolamin~ pH 9.0
for 1~ hour6 at 4~c. The gel then wa~ wa~hed with three
cycle~ of alternating pH. Each cycle c~nsisted of 0.1 M
acetate huffer pH 4.0 contalning C.5 M NaCl followed by a
iO wash with 0.1 M Tris-HCl pM 8.0 conta~ning 0.5 M NaCl.
The coupled phosvitin-Sepharo~e was s~ored at 4 to 8~C in
100 mM Trig-HCl pH 8.0 containinq 0.5 M NaCl and O .02 %
~odium azide and used for the Examples described below.
Example 3
Illu~tr~tion of the bindin~ ca~acity of the ~o~vitin-
Sepharose chromato;TraDhic,~ gent
1 ml bed volume columns of the phosvitin-SepharoQe
chromatographlc agent were u~ed, which had been
equilibr~ted with 50 mM Tris-HCl at ~ pl~ 0~ 7.5.
To one s~lch column, o. 25 mg o cytochrome-C
(obtained fro~ horse heart~ dissolved in the
equilibrat~ng b~fer was l~aded. The buffer wash showed
negligible material ab30rbing At 41~ nm ~soret ~n~ o~
cytochrome C). The protein was eluted from the ~olumn
with 20 mM sodium phosphate buf~er pH 6.5. The recove~y
in th~ .-luted sample was more than 90%.
17(~6 ~ 12 ~71 ,?,4- yg~g ABEL ~ IM~A~ 2 0 4 ~ 717 ~ 23
~ 20 -
In order to ohe~X the specificity, cytochrome-C was
nllowed to react with ~oluble phosvitin and then loaded
on to the pho-~vitin-Sepharose. The loaded miXtUre did
not bind to the colu~n clearly suggesting that the
bindiny domain on the cytochrome-c i6 already masked by
phosvitin.
~ hi~ e~tablished the efficacy of the colu~n
material, viz. phOsvitin.
&~3~e 4:
~ of lysozyme f~om e~ whi~e:
A phosvitin-Sepharose 4~ column (5 ml bed volume)
was ufied for thi~ purpo~e. Egg white was diluted in
mM KH2P04 pH 6.24 to ad~ust the OD220 to approximatelY 10
per ml. A total o~ lo ~1 of this diluted egg white wa~
directly l~aded on~o the phosvitin-Sepharo6e, previously
equilibrated with 66 mM KHzP04 pH 6.2~. The ~low rate
was maintained at 1 ~1 per 3 min~te~ u6ing a peristaltic
pump. After the loading was complete, the column wa~
washed wi~h 66 mM KH2P04 pH 6.~4. When the A280 reading
was below 0.05 OD, the column was eluted with 100 mM
X~2P04 pH ~ . 24 containing 200 mN NaCl. ~he fractions
(lml vol) were monitored for A~80 absorbance ag well as
enzyme activity.
The enzyme activity wa~ measured in 66 mM K~2P04 p~
6.24 containing the requisite amount of MicroçQccus
luteus cell suspension at 25C. One Unit is defined here
7~n~ g1 1~ 13 ~(171 ~2 ~ ABEL & IMRA~ 2 0 4 4 71 7
- 21 -
a~ the decrease in t~e optlcal density of Q.l/min. Rt
450 nm under assay ~onditions.
The total loading in term~ of enzyme unit w~s 4300
~ith a 6pecific activity of 3~.6 units per mq protein.
~he salt elut~on ~ave a recovery of over 70% with a
specific activity in the range of 420 and above. The
three times recrystalli6ed preparation from Sigma under
identical conditions gave a spec~fic activity of 437.
The enzyme preparation wa~ analysed by ~odium
dodecyl~ulphate polyacrylamide electrophoresls and found
to contain i~ addition to lysozyme a 6mall amount o~
ovalbumin which ha6 a molecular weight of 45,000.
I~:xam~le
Blnding of ECoRX ~nd Bam HI to Pho~vitin-Sepharose
~00 units o~ each of these r~striction enzymes
obtained from Bangalore Gen~i Company were loaded onto
separate columns of phosvitin-SepharoQe 4s, whi~h had
been equilibrated with lO mM Tris-HCl pH 7.5 containing
50 mM N~Cl, 5 m~ MgS04 and 1 m~ DTT. These enzyme6 were
eluted with lO mM Tris-HCl pH 7.5 ~ontaining 1.5 M NaCl,
10 mM KCl, lO0 ~/ml BSA a~d lm~ D~T. When t~e salt
con~entration was l.0 M, there wa8 no enzyme elution.
The a~ay was based on l~nesriz~tion of purif ied PUC8
plasmid and gub~equent electrophoresis of DNA on 1%
aga~ose gel in Tris-Borate ~DTA buffer 6ystem.
17f~ 1 19 14 ~)71 ~4~ 9~ REL & IM~ 20~717 ~025
-- 22 --
~X~
FSH binding and elution
Pho~vitin-Sepharose 4B column (0.~5 ml bed volume)
wa~ used. The column was eq~ilibrated with lo mM Tris
s HCl pH 7.75. The column was loaded with human Follicle
Stimulating Hormone (corresponding ~o 7 units/l). ~he
column wa6 waghed with the equilibration buffe~ and 8
fractlons of 0.25 ml were collected. The column w~s t~en
eluted with lOmM ~ris HCl p~ 7.75 containin~ lM NaCl and
8 fractions of o. 25 ml were co~lected. The fractions
were subjected to Delfla ~ as~ay. FSH wa~ quantitatiYely
recove~ed in the third fraction of the el~tin~ buffer.
~x.ample 7
Aff~nity_of_ ho6vit_n-Seph~Fo~e to Adrenocorticotroplc
Hormone ~1~1
Phosvi~in-sepharo6e (0.25 ml bed volume~ was
equilibrated with 10 mM T~is H~l pH 7 . 7. 2~0~1 of ACTH
(human) 1 mg/ml wa~ loaded on the column and the column
washed with the same buffer. The column was flr~t eluted
~o with 10 m~ Tri3 HCl pH 7.7 containing 1 M NaCl. The wash
and salt eluate6 were assayed for AC~ by radloimmun~-
assay. It was obherved that ACTH is bound to the column
and i~ eluted by 1.0 M NaCl.
17io~sl 19 15 ~)71 ~2 ~(S~ ABEL & IMRAS! 2044717 r~ a
- 2~ -
~xa~le 8
L4-68
Phosvitin-sepharose (0.25 m~ bed volume~ wa~
equilibrated with 10 mM Tris Hcl pH 7.7. 200 ~1 of
parathyroid hormone (PTH) ~44-68) R72 pmole/l was
loaded. The column was first eluted with lo ~M Tri~ ~C1
pH 7.7 containing 1 M NaCl and the second elu~ion was
done with lo mM Tris HCl pH ? .7 containing 1. 5 M NaCl.
lo Afisay for the wa-~h an~ salt eluates wa~ done by
radloimmunoassay. It was observed t~at no elution was
obtained with 1 M NaCl, and 1.5 M NaCl wa~ required for
eluting PTH from the phosvitin-Sepharose col~mn, indicat-
ing 6trong bindinq.
It is clear from Example~ 7 and 8 ~h~t the
pho-~vitin-sepharose al~o binds to hACTH and ~P~H ~rom
sexu~ -~amples.
EXAMPLES WITH MODI~D P~OSVI~
Exam~le 9
20 Modification of Phosvitin
Phosvitin was modif ied by p~oteoly~is of the nativ~
protein with trypsin as follo~s:
~rypsinisation:
Phosvitin (purified on the Mono Q column on the FPLC
17~0~ 16 ~071 24~ A~EL............ ~ 1~1RA~' 2044717 ~027
- 24 -
~y~tem) wa6 taken up in 20 mM Tris HCl pH 7.5 at a con-
oentration of 20 mg/ml and wa~ treated with trypsin (in a
phosvitin:trypgin rAtiO tW/W] of l00:l). The mixture Wa8
incubated at 37C for 1 hour. On SDS-PAGE the native
protein showed 3 ma~or bands around 28-32kDa. The
trypfiinised pho~vitin ~howed a major band at ~6 kDa and
two other~ corre~pondinq to molecular welght~ o~ around
8000-14000.
Purification of trypsini~ed pho~vitin:
The trypsinised phosvitin was -~ubjected to Cu(II)
imino di-acetic acid, metal chelation chromatography.
For this purpo6e chelating Sepharo6e fast ~low column
(l ~l, Pharmacia) was eq~ilibrated with a solu~ion of
50 mM copper ~ulphate till th~ ~ntire column wa~
~oloured. The column wa~ then equilibrated with 20 mM
sodium pho6phate buffer pH ~.5 containing 0.5 M NaCl and
~hen it was loaded with 200 ~l (2 mg) protein solution.
The col~mn was developed with a p~ gradient generated
using 20 mM ~odium pho6phate buffer pH 7.5 containing
0.5 M NaCl (buffer A) and 200 ~M sodium pho~phate buffer
pH 3.5 containlng 0.5 M NaCl (buffer 3). All operations
were performed on the FPLC 3ystem (Pharm~Cia) at 21C.
Some A280 ab60rbing material ~i.e. that absorbing at
2~0 n~) (peak Pl) wa~ eluted in the buffer A wash and
later, at the end of t~e gr~dient, the pH wa~ continued
to be ~qin~ained at 3 5 when another protein (p~ak P2
was ~bt~ined. Analy~iS of these peak~ on SDS-PAGE
17 l)~ Y1 19 17 ~()71 '4~ ~Y~9 ABEL ~ IMRA~ 2 0 4 ~ 71 7
- 2S -
confirmed tha~ peak P2 represent~ a truncated version of
phosvitin which correspo~ds to a molecular weight of
Z60~0.
Analytical ~esults:
Althoug~ the molecula~ weight of truncated phosvitin
wa6 less by abou~ 4000-5000, oompared to the native
protein, the~r phoqphate and ~arboh~drate content
remained essentially sl~ilar.
Immobili~ation-Qf tr~ncated phofivit~n
The P2 ~ractlon was dialysed again~t 5 mM EDTA and
then against ~illi Q water. The dialysed protein was
lyophil~zed, and covalently attached to CNBr-activated
Sepharose (protocol of couplinq was the ~ame a~ described
1~ for phosvitin-Sepharose). 1 ml of coupled ~atrix
typi~ally contain 6 mg prote~n.
T~e coupled matrix wa6 de~ignated as P2-Sepharo6e
and wa~ te~ted for its binding ~haracteri~ticS as
de~cribed below.
Ex~ple 11
The m~trix (0.5 ml bed volume) was equilib~ated with
ZO mM Tri~ HCl p~ 7.5. T~e column was loaded with 200 ~1
of 1 mg/ml of horse heart cyrochrome-c ~Sigma). The
z5 column was washed with the equilib~ating buffcr till AZ80
.. ~
I
17~n6 ~ 7~ 3~ ABEL & I~RAY 2 0 ~ 4 717 ~02~
- ~6 -
was nearly zero, and then eluted with 20 m~ Tri~ HCl pH
7.s containing 1 M NaCl. The salt-elut~d fractions
showed the soret band at 410 nm. The sodiu~ dithionite
reduction which r~6ulted in the appearance of ~, ~ & Y
band~ confirmed the presence of cytochrome-C.
The binding characteri3tlcs of cytochro~e-C to P2-
Sepharo3e were very similar to tho~e of pho~vitin-
Sepharo6e matrix.
Example 12
lo Affinitr of ~ysozrme to P2~SeDha~ose
The 0.5 ml bed volume of P2-Sepharose column wa~
equilibrate~ ~ith 20 mM Tris HCl p~ 7.5. The column wa~
loaded w~th 500 ~1 of 14 mg~ml lysozyme ~Sigma). The
matrix wa6 washed with the equilibration buffer till ~.e
A280 was zero, a~d then eluted with 20 mM Tr~s ~Cl pH
7.5 con~aining lM ~aCl. Virtually no A280 absorbin~
material appeared either in the ~reakthough or ln the
wash ~ractions. There was es~entially total recovery
(>85%) of ly60zyme in the salt-eluted fractions. The
~o pre~ence of lysozyme in the eluate W~G con~rmed by
subjecting the fractions to enzyme aE;~iay. P2-Sepharo~e
has a ~apacity to bind to 13-14 mg of ly60zyme per ~1 of
gel bed volume, whereas ~he equiv~len~ capacity for
phosvitin-sepharose i~ 5-~ mq/ml.
The column wa~ u~ed for puri~ication ~f ly~ozyme
from egg wh~te. The crude egg white prepa~ation was
I7~ 7I '~Jy~.~ ABELI~ IMRA~ 2 0 ~ ~ 71 7 ~30
- 27 -
diluted to l0 OD (A280)/ml with 20 mM Tris HCl pH ?.5.
The P2-Sepharo~e column (l ml) was loaded with 50 OD o~
crude protein. The breakthrough p~otein did not ~how
any apprecia~le lyso2yme activity, whereas the 20mM Trls
HCl pH 7.5 containing l.O M NaCl eluates ~howed very hig~
specifi~ acti~ity.
In one experiment 7.5 mg of ly~ozyme could be
purified with ~pec~fic activity of 42~ u/mg protein (Unit
definition: 1 unit of ly60zy~e causes A450 change of
0.l/min at 2SC at pH 6.24).
~xa~ple 13
Purificat ~ oRl on P2-SeDhaFose
EcoRl wa~ purified from t~e extract of Ltrain RYl3
of E. ~o.li. The extract of ~ coli (RYl3) wa~ prepared
accordinq to ~he following p~otocol:
T~e strain was gro~n on L-broth pH 7.0 ~tryptone
l0g/l; NaCl l0 g/l: Glucose 5 g/l and Yeast extract
5 g/l), till the OD660 was l.0 to l.l. ~he cell6 were
harve~ted by centrifug~tion at l0000 rpm for l0 min and
washed in TEM (20 mM Tris HCl pH 7.5 contain~ng 2~M EDTA
and 1 mM B-mecaptoethanol~. The cell6 were taken Up in
TEM and lysed ~y sonicati~n for l0 min u6ing Sonics and
~ater~al; Vibra ~ell Microtip. The lysed ext~act was
centrifu~ed and the 6upernatant was treated with 5%
~final c~ncentration~ ~treptomycin sulphate ~t 4C for 45
min. ~he ~olution was centrifuged at 15000 rpm fo~ 15
17~ 2~1 ~il71 ~? ~ EL ~ I~IRAY 2 0 ~ ~ 7 1 7 ~31
- 28 -
min. and the supernatant W8B dialysed again~t TEM for 24
hour~. The dialy6ed extr~ct had typically 8.0 mg~l
protein.
1 ml P2-Sepharo6e wa~ equilibrated with T~M buffer,
and 2 ~1 of the extract was loaded on the column. The
column wa~ washed with TEM til l the A280 was zero and
then eluted with TEM containing l.S M NaCl. To the peak
fraction~, ~SA (bovine ~erum album~n) w~ added to a
final concentra~on of 100 ~g/ml an~ the peak fractions
dialysed against TEM overnight and a~sayed for EcoRl
activity.
We have used lambda-DNA digestion a~ay to detect
and quantitate the EcoRl act~vity. We can purify
typically 5500 units of EcoR1 usin~ 1 ml bed volume o~
P2-8epharo~e.
