Note: Descriptions are shown in the official language in which they were submitted.
2 ~
BEHRINGW~ TIE~3Gl~SE:Ll,SC~lA~r HOE 8g/B 046 - ~la 796
Dr. Lp~rd
D~scription
A process for the expression of forei~n ~enes Ln yea~t~
The invention relates to the optimization o~ the expreR-
~ion of foreign genes, especially reoombinant f~ctor
XIIIa [rFXIIIa) in yeas~ he optimization wa8 e~en-
tially carried out by use of a hybrid GaluAs /CycI
promoter, deletion of the 5' non-translated region and
truncation of the 3' non~transla~ed region of the FXIIIa
: cD~A, selection of specific yeast strains and a par-
ticularly sui~able growth medium compo~ed of whey or whey
hydrolyzate. Quantities achievable in thi~ way are
2 IOO mg/l FXIIIa in shake cultures and ~ 500 mg/l in
fermentation after scale up.
Clotting factor XIII (FXIII) forms the final member in
lS the coagulation cascade in the ~tural process of blood
clotting. FXIII was described as a ~erum factor for the
first time by Robbins in 1944. Robbins had observed that
fibrin clots from normal whole blood are in~oluble in
urea solution whereas clots from highly purified fibri-
: 20 nogen and thrombin are ~oluble.
. .
The molecular w~ight of FXIII from pla~ma is about
300 kd. The plasma protein i~ composed of subunit A of
about 80 Xd ~nd subunit b of about 75 kd and e~i~t3 as
a tetramer wi~h two a ~nd two b subunits t~2b2). Sub-
unit a tFXIIIa~ contain~ the enzymatic activity. FXIIIa
i activated by elevage of an ~nino-tenminal peptide
which is 4 kd in ~i~e by thrombin ~o ~ive FXII~a'. This
activated ~orm o~ FXIIIa act~ ln the presence of calcium
~ ions as a kran~glutamina~e and preduces cro~linked, via
`;1 30 intermolecular ~-glutamyl~~-ly~ine linkage~, fibrin
monomers to give fixmer clots.
The cDNA and the first instancas of expression of FXIIIa
'`" . :
.-~. . . :
:;.: . . .. . .
: :- . . . .
2~3~81~
in E. coli, animal cells and yaa3t~ iB described in
EP 0,236,~78. Th0 yield~ o~ active FXIIIa in yea~ts are,
however, rather low, at 150 ng/ml, in the process de~
cribed.
:
FXIIIa can be expre~Red in high yields in E. cDli (Amann
at al., Gene 69, 301-315 [1988]). However, ~oBt of the
FXIIIa synthesized in E. coli i~ in~oluble and thus not
- enzymatically active. A suitable alternati~e to hetexo-
logous e~pression in prokaryote~ i~ the synthe~i~ in
eukaryotic cells. Heterologou~ expression of biologically
- active FXI~Ia in CHO cell~ ha~ already been demon~trated
(Zettlmei~l und Rarge~, (19B9) r XIIth Consress of the
: International Society on Thrombos~ 8 and Haemo~tasis,
Tokyo). Nevertheless, the yields in this ca~e are inade-
lS quate to ensure cost-efficient production.
Yea~ts are suitable as host cells be ause, on the one
handl their genetics have been well characterized for
molecular biological operations/ cloning and expre~sion
vectors are available and~ ~s eukaryotes~ they poS8e66
the typical post-translational modifica~ion machanisms.
On the other hand~ as microorganisms, they have a rather
short generakion tLme ~nd there has been aufficiant
development of fermentation procasses for cult~ring
~ yeasts in high cell den~ity. It ha~ already been ~hown by
:: 25 way of example in European Patent Applic~tion
EP A2 0,236,978 that bioloyically actiYe FXIIIa can be
prepared in baker's yeast. Recombinant active F~IIla can
also be synthesized in the fi~sion yeast
: . Schizo~accharomyces pombe with the aid of molecular
biolo~ical techniques (Br~ker ~nd ~uml, F~BS Lett. 248,
105-110 [l9B9]). ~owever, the yield in the two examples
described il3 1 at 0.15 and 2 mg/l resp2ctivaly, no~ in a
:~ range which make~ preparation appear economic. Higher
yields are described in the EP Application A2 0,268,772.
Plasmids in which FXIIIa ~ynthesis i~ under the control
of the strong glycolytic TPI promotar result ~n a
synthesis rate of 10 mg/l. 50 mg/l are achieved usin~ the
~:.
.. . - ; :
;
.,
.
2031810
-- 3 --
ADHII promoter.
~ence the ob~ect was to improve the expres~ion of FXIIIa
so as to achieve values 2 100 mg/l. With such expre~sion
rates the genetically engineered prepar~ion of FXIIIa i8
more cost-effi~ien~ than cla~sical purification from
placentae.
We have found tha~ the use o~ a hybrid GaluAs /CycI
promoter, the deletion of the 5' non-tran~lated region
and a truncation of ~he 3~ non-tr?n~lated xegion of
FXIIIa cDNA in the expre~sion vector, the ~slection of
specific yeast strains and the U50 of a very suitable
growth medium containing glucose and galacto~e alone or
in combinati~n result in a high yield of F~IIIa. The
: hybrid Gal uAs~CycI promoter i8 derived from pENBLyex4
: 15 (Cesareni, G. und Murray, A.H. in Se~l~w, J.~. (ed.~
Genetic Engineering, Vol. 9, 135-154, Plenum Publi~hing
;~ Corporation, 1987).
. . ;, .
: Representative vector~ con~ain the FXIII cDN~ without the
5~ non-translated and 3~ non-translated cDNA region, the
latter preferably ~hortened from 419 base pairs (bp) to
only 120 bp. The synthesis of the specific vectors pMB307
and pMB330 is ~hown in the f igure and in the e~mples.
The fund~mental basic vector i~ the above ~entioned vector
pEMBLyex4 in whose polylinker the above mentioned
~runcated FXIIla cDNA has been ligated.
pEMBLyex4 is located episomally in yeast in a copy number
of abou~ 50/cell and carrie~ ~he 8e1QCtiOn markers Leu2d
; and ~ra3, so that complementation of yeast strains with
: the genotype leu2 and ura3 iB pos~ible; complementation
of Trpl ~trains al80 occurs on insertion of the Trpl g~ne
(see Example 2) in p~B307 to give p~B307T.
.~
Most ~uitable for p~B307 were the S. cerevi~iae strain~
AH22 and Cl.3A~YS8S, and for p~B307T the ~train 150-~B.
AH22 i~ described in A~ Hinnen et al. (Proc~ Natl. Acad.
~?'`:','
: ~,,':: :, ',' :, ' " ' , ' ''
'.' ' ~ ' ' ,
,~'' : '.
. ~, ,~, ,
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2~31810
Sci. USA ~1978) 75, 1929-1933); C13~B~rS83 ha3 been
di~clo~ed in EP-Al-0, 327 " 797 " and 150-2B has been des-
cribed by C. Baldari et al. (EMB0 Journal, (1983), 6,
229-234L) .
The presence o~ glucsse in the mQdium repre~e~ ~he
induction of the Gal or Gal/u~s promo~ers by galacto~e.
Surprisin~ly, the ~ynthe~is r,ata increase~ v0ry greatly
when galacto~e i al~o pre~ent in the medium in addition
to glucose from the ~tart of the fermentation onwards.
Hydrolyzed whey powder i8 e~pecially ~uitable and,
moreover, is obtainable at low C08t by compar1son with
pure galacto~e. An alternative i~ whey powder with added
~-Galacto~idase or lactose-cleaving microorganisms.
~ccordingly, the invention relates ~o the uPe of hydro-
lyzed whey powder in conjunction with yea~t expressionplasmid~ under the control of a galactose-inducible
promoter (1), preferably the hybrid Gal uAs/CycI promoter
(2), and preferably the pEMBLyex4 vector framework into
which the FXIlIa cDNA from which the 5~ non-tran~lated
region has been deleted and the 3t non-tran~lated region
has been truncated hax been in~erted (3) is us~d.
Preferred 6trains for the expression are the ~trains AH22
t4) and C13ABYS86 (S) for Leu or Ura ~election and 150-2B
for ~rp selection (6).
The combination of the abovamentioned features (1), t2),
~3), (5) and (6) i~ particul~rly preferred ~or FXIIIa
expression optLmization. On implementation on the indu8-
trial scale in 100 1 or larger fermenters, F~IIIa con-
centrations 2 500 ~g~l were achieved with the ~bove-
mentioned proce~s.
The invention i8 ~urther described in the examples andthe patent cla.ims.
. ~ , . .
;" " '''
.':.~ ' :
203181~
-- 5 --
~xam~les:
The molecular biological techniques u~ed herain are
mainly based on ~aniatis et al.: Molecular Cloning. A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold
Sprinq Harbor (1982) and, w:ith regard to the work on
yeasts, e~pecially on Rodri~lez et al.: Reco~binant DN~
Techniques. Addison Wesley Publishing Company, London
(1983~. The hosts used for the ~nthe~i~ of FXIII~ were
generally accessible laboratory strain~ of baker'~ y~ast
Saccharomyces cerevisiae which have one or more defects
; in bio~ynthetic enzymec. The~e defects are complemanted
: by the corresponding functioning genes on the vector~ in
each case. The yeast strains were transformed by the LiCl
method as has been optimized for the fission yeast
(Broker, ~., Biotechniques 5, 516-518 [~987]), with the
modification that the heat shock was carried out at 42C,
not at 46C. Single clones can be selected on YNB minimal
medi~m three to four days after the transformation with
~ plasmid DNA. ~he culturing of the tran~for~ant~ in liquid
- 20 culture to obtain FXlIIa was carried out as follows:
~ 50 ml of YNB medium in a 250 ~1 Erlenmeyer ~lask with
.~ baffles on the side were inoculated with a ~ingle colony
and shaken at 30C for two days. When a yeast ~trai~
whose totality of genstic defec~ was not complemented by
the vector was used, 0.02 0.04 mg/l of the appropriate
amino acid or of the base wa~ added to the medium. 10 ml
of ~uch a preculture were u~ed to inoculate 100 ml of
. complex YPD medium. The 108s of plasmid under the~e non-
~elective conditions varied with the plasmid and yeast
strain used. Cultures in which FXIIIa synthesi~ i8 under
.~ the control of the Gall or GaluAs /Cycl hybrid promoter
which can be regulated by ~alactose were mixed with 2%
galacto e 24 hour3 after the inoculation, in order to
induce the Gal pro~oterO
; 35 Baker's~ yea~t is able to grow on medium containing
: ~alacto~e as the ~ole carbon ~ource. Galactose is trans-
ported via a specific ~alactose permease into ths cell
~,. '''
:
203181~
-- 6 ~
and finally enters glycolysi~ as glucose l-pho~phate. The
following enzymes are involved in thi~: Galac~oki~ase
(Gall), D-Galactose-l-phoRpha~te uridyl~ran~ferase (Gal7)
and uridine-diphosphogIucos0 4-eRimerase ~allO~. The
genes of these emz~mes are clssely ad~acent and sub~act
to coordinated regulation. When the carbon source i8
changed from glycerol or glucose to galactoBe~ tr~n-
~cription of the~e genes ig enhanced 1000-~old. In the
absence of galactose, Gall, Gal7 and GallO are repre~sed
by the nega~ive regulator protein which i8 encoded b~ the
Gal80 gene. A po~itive regulator protein which is sncoded
by the Gal4 gene i~ required for the expre~sion o~ Gall,
Gal7 and GallO. There i~ con titutive synthesiR of amall
amount~ of Gal80 and Gal4 protein~. The Gal80 protein
binds to Gal4 protein and, in this way, inhibits
activation of the genes by the Gal4 protein. ~n inducer
(galactose or a derivative of this sugar~ presumably
binds to a site on Gal80 80 that the affinity of the
Gal80 protein $o the Gal4 protein i~ reduced and Gal4
becomes free. Gal4 now pre~umably binds with it~ amino-
terminal end to the 5~ region~ of Gal 1, Gal7 and GallO
and brings about the enhanced transcription of the gene~.
These distal DNA regula~ory elements of yea~t genes are
called upstream activating sequences (UASs).
xample 1: Synthesis of the plasmid pMB307
The ~tarting material i~ pFXIII 104 (~mann et al.,
~ehring Inst. Mitt. (1988), 82, 35-42; Fig. 1) and
p~MB~yex4; the plasmid pEMB~yax4 i~ ~n E. coli ~huttle
vector. ~he e~Eential relavant properties of tha vsctor
are that they can be selected in E. coli on the b~sis of
the ampicillin-resistance gene. In yeasts, ~alection
~akes place via the Ura3 or Leu2-d gene in leucine-
and/or uracil-auxotrophic ~train~. ~table replication in
yeasts i6 en6ured by the content of 2~ DN~. A particular
: 35 feature of the vector is the Gal~Cyc hybrid promoter.
~enes can be in~erted into the polylinker at the 3' end
of this element so that, in yeast~ with a recombinant
. `; ' . I
:, ` " , '
' ` , ' ' ~ '
203181~)
plasmid of thi~ type, expressi.on of ths forei~n gene i~
under the control of ~he ~al~Cyc promoter which can be
regulated. Efficient transcript:ion termination ie ensured
by an appropriate DNA unit whic:h i8 located at the 3' end
of the polylinker. The construc:tion of pMB307 i~ dep1cted
diagrammatically in Figure 1.
., .
A pla~mid with deletion of the 3~ non-tran~lated re~ion
beyond 120 bp did n~t achieve any ~urther increase in
expression.
10 Example 2: Synthesis of the plasmid pMB307T :
The plasmid pMB307 carries, as ~election marker~, the
:: genes Leu2-d and Ura3, which are usad to complement
: strains which have the genotype leu2 and ura3. Trans
formation with a modified p~B307 whi~h carrie~ the ~rpl
gene has the advantage that the pla~mid 1088 occurring in
~trains with the g~notype trpl in c~mplex media which
contain acid-hydrolyzed protein~ as nitro~en ~ource i8
very low. The amino acid tryptophan is easily decompo~ed
by acid a.nd i6 no longer present in ~uch media ~o that a
complex medium itself xepres~nts ~ selectiv~ m~dium with
respect to the Trpl marker.
The vector p~B307 was linearized with ~t~ nd the
StuI/EcoRI DNA fra~ment, which is 830 bp in size and
carries the Trpl gene, from the plasmid pGT2 (D~, S.,
Rellermann, E. and Hollenbsrg, C. J. ~teriol. 153,
1165-1167 ~1984)) was ligated in. The new pla~mid p~307T
i~ now able ~180 to comple~ant trpl str~in~. V~rious
~train~ wer~ tranafor~ed wi h pM~307r, and the ~IIIa
yield wa~ determined~ ~he ~train with the highe~t FXIII~
~ynthe~is rate ~mong the ~ested ~trains w~ 150-2B.
Exam~le 4~ 5ynthesi~ cf the pla~mid pMB330
,.
The FXI~Ia cDNA mentioned in EP 0,236,978 code~ in
position 88 (CTC) for ~he amino acid leucin~ hinose et
,"~ : ~ .. '''' ' '
.
.
:: ,. . .
. . .
;,': :. ~
~: , . .
.
~' ,
2~3~810
al. ~1986), Blochemistry 25, 6900-6906, by ~ontra~t,
found a TTC triplet which code~ for phenylal~nine in this
po~ition. The amino-acid ~equence of FXIIIa, which has
been determined by Takahashi et al. (1986), Proc. Natl.
Acad. Sci. USA 83, 8019-8023 likewise revealed a phenyl-
alanine at position ~8. It is thus possible that the
amino acid leucine deduced in p~iti~n 88, which i~
mentioned in EP 0,236,978, is a relatively rare allele by
comparison with phenylalanine. ~ FXIIXa expre~sion vector
(pMB330) which has exactly the same structure as the
plasmid pNB307 and differs only in that the CTC codon has
been replaced by a TTC codon, ~hat is to ~ay code~ for a
phenylalanine in position 88, wa~ tharefore constructed.
Example 5: Effect of various strains on the FXIIla
synthesis rate with the plasmid pMB307 or
: pMB307T
Plasmid pNB307 en6ures in S. cerevisiae 252 a ~XIIIa
sy~thesis of about 5 mg/l under the optimized culture
conditions. The yield of FXIIIa in the strain HD was
considerably less. This indicates not only tha~ a high
- FXIIIa synthesis r~te depends on the plasmid and medium
but also that cellular factors are also able t~ influence
the yield. 5everal strains were therefore separately
: transformed with the vector pMB307 and analyzed for their
capacity to Mynthesize PXIIIa in shake cultures. The
: result i~ summarized in Tab. l. It emerged that it i~
possible to achiev0 a wide variation in F~IIIa yields
with the individual strains. It w~s poasible to ~chieve
high FXIIIa ~ynthesis only in strains which reach ~ high
~ell density and in which pla~mld 10~8 i~ low. On the
other hand, a strain which grows well ~nd h~3 ~ low
plasmid 108s does no~ necessarily ensura a ~a~isfactory
yield of F;KIIIa. A~toniRhingly, the capacity do~8 not
correla~e with few auxotropic marker~ either; this is
becaus~ even stxains with three or aven five defects in
bios~nthetic enzymes achieve cDnsiderable yields of
5-7 mg/l FXIIIa. ~oteworthy amon~ ~he tested ~trains are
,~ . . . .
~:~ . ' . : . .
2~31810
g
the strains AH22 and C13A~YS86 with yields above 10 mg/l
FXIIIa. The plasmid pM~307 carries, as selection marker~,
the genes Leu2-d and Ura3, which are used to complament
strains which have the genotype leu2 and ura3. Tran~-
formation with a modified pMB307 which carrie~ the Trplgene has the advantage ~hat the plasmid 108~ occurring in
s$rains with the genotype trpl in complex media which
contain acid-hydrolyzed proteins as ni~rogen BOurCe ia
: very low. The amino acid tryptophan i8 ea~ily decompo~ed
by acid and is no longer present in euch media ~o thst a
; complex medium it~elf repre~ents a selecti~e medium with
re~pect to the Trpl marker.
The vector pMB307 was linearized with BstEII, and th~
StuI/EcoRI DNA fragment, which is 830 bp in ~ize and
caxries the Trpl gene, from the plasmid pGL2 (DasO S.,
Kellermann, E. and ~ollenberg, C. J. Bacteriol. 158,
1165-1167 tl984)) was ligated in. The new plasmid pMB307T
is now able also to complement trpl ~rain~. Various
strains were transformed with pMB307T, an~ the FXIIIa
yield was determined. The strain with the hishest FXlIIa
synthesi~ rate among the tested strains was 150-2B, with
8 mgtl PXIIIa.
.
:: Example_6: Effect of the medium on the F~IIIa yield in
pMB30~ tra~6formant~
.~
Starting from the yields which were achieved
using the plasmid pMB307 in shake culture~ in cu~toma~y
media, attempts were made to increase tha synthesis rate
by altering the componont~ o~ the media. The tran-
scrip~ion of the PXII~a-~pecific cD~A in yeast cells
which carry the plasmid p~B307 i9 regulated by the carbon
seurce. No P'XIIIa i~ produced in media which con~ain only
gluco~e. ~ests were also carried out to find whether the
timing of indu~tion of FX~IIa-~pecific ~ran~cription has
an effect on the FXIlIa yield and whether the ener~y
source glucose can be replaced during the gxowth phase by
other carbon asurces without reducing ths yields
.
, . .
' ' .:
.': ' ~ ' ' .
~' .. .
203181~
of FXIIIa.
Wh~n the glucose in the ~PD medium i8 replaced by maltose
or glycerol, and the rultures are fed with 2~ galacto~e
after 24 h, once again the yields are about 2.5 to
3 mg/l. ~y contrast, when glucose in the medium is
:. replaced by galactose from the ~tar~, ~he yield of F~IIIa
is only about 1 mg/l. This relativaly low yield 18 not
: attributable to reduced growth, because the cell number is
about SxlO~/ml which iB exactly a~ hlgh as in culture~
which contained only glucose or were f ed with galactose
. after 24 h. Presumably FXIIIa syn~he~is has an adverse
:. effect on cell metaboli~m ev n during the growth pha~e
and, in this way, Lmpairs FXIIIa ~ynthe6is during the
idiophase. This observat.ion is ~upported by the fac~ that
the yield of FXIIIa likewise does not exceed 1 mg/l in
: shake cultures in which FXIIIa synthesis is under the
- control of the strong but constitutive ADC promoter.
The yield of FXIIIa in yeasts can be increased to 4 mg/l
by not only inducing FXIIIa ~ynth~sis by adding galacto~e
after 24 h, when glucose is no longer present in the
medium, bu~ also adding ~alactose once again, after a
further 24 h and 48 h, in a final concentration of 2% in
:~ each case. When, at the ~tart of the fermentation, the
complex medium co~tains not only glucose but also galac-
tose in addition, and when feeding with galacto~e is
- carried out each day, the production rate even increas2s
above 5 mg/l~ This supply of the YPD medium with glucose
and galactose i callad YPDG medium hereina~ter. It
: appear~ that FXIIIa synthesis i~ not induced ~y galactose
:~ 30 as long as glucose is pre~ent in the medium. On ~he other
handl under these conditions, the metabolic ~ha~ge-over
after cons~ption of the gluco~e can take place imme
diately, and in the late qrowth phase galactose actq both
as energy ~ource and as inducer. Thu~, it was possible
with a suitable composition of the medium for the yield
of FXIIIa to be approximately doubled by compari~on with
~he cu~tomary standard m~dium.
: .
.. . .
:;' '
~; .
2~3~810
.. 11 --
Exdmple ?- Whey and whey hydrolyzate as ~ource of galac-
tose
~:'
Owing to the vector~ pMB307 snd pMB330, FXIIIa synthe~isi6 strictly repres~ed by glucose in the medium and i~
S induced by galactose. It i~ evident that as long as
glucose i3 present in the medium induction i8 prevented
even in the pre~2nce of galacto6e. Since galacto~ is a
c08tly sugar, attemp~ were made to bring about induction
not by pure galactose but by a reasonably priced medium
which contain~ galac~o~e among~t other thin~. Hydrolyzed
whey powder from Molkerei-Zentrale Wes~falsn ~ippe eG
(Munster) was te~ted for this purpo~e. Thi~ whey powder
contains, apart from protein, fat and mineral~, about 18
glucose, 13% galactose and about 23% lactose too.
20 g of hydroly~ed whey powder were stirred in 100 ml of
water, heated to 60C and thus dissolved. ~his prepara-
tion w~s centrifuged at 10,000 g for 30 minute~ and then
the supernatant was filtered ~terile. Thi~ solution was
dilu~ed either 1:1 wi~h sterile water (A) or 1:1 with
concentrated YPD (B~. The FXIIIa yield with ~. eerevisiae
C13A~YS86 tpMB307 or pMB3303 was only about 5 mg/l ~n
medium A. By contrast, up to more than 100 mg/l F~IIIa
was detected in medium B. I~ was thu~ po~ible to 8how
that whey powder, vf which an exce6s is produced in
dai~ying and which i8 thus very r~a~onably priced~ can be
employed, ater h~drolysi~ of the l~ctose content to
glucose and g~lactose~ as a suita~le ~ubstance for the
recomb~nant ~ynthe~i~ of FXlIIa. In fdCt, ~ubstances
which have not yet been identified result in n con-
siderably higher FXIIIa synthesis compared with culture~which have been i~duced with pure galacto~e. It wa~ al30
pos~ible to achiev~ the high FXIIIa yleld~ ~hen ~ par-
tially dsmineralized condensed cheese whey from De Melk-
industrie Veghel ~BA Veghel~ NL) wa~ employed. Another
sui~able source of ~alactose a~ an alternative ifi whey
powder which, untreated, contains lacto~Q but no galac~
tose. Thi~ lacto~e present in whey powder was hydrolyzed,
".. ~ . . . . . .
.~ ,...... ~
-,.. . .. ~ ,
;:
. .
2~'31~10
- 12 -
af~er the pswd~r had been suspRnded in YPD, to glucose
and aalactose with ~-Galacto3idase from E. coli. A
pr~paration of this type also brought about high F~IIIa
synthesis in yeasts. It was even possible to add ~-
Galactosidase from E. coli o:r Kluyveromyces lactis (infree form or immobilized) to 1he ye~st culture which had
been supplemented with whey powder uo that galacto~e wa~
liberated by cleavage only du:ring the farmentation.
Also suitable for ~XIIIa production in place of whey
hydrolysa~e a~ source of alacto~e is lackose hydroly-
s~te. Commercially available lactose hydrolysate (for
example Biolac GmbH, D-3221 Harbernsen or IMA GmbH,
D-6078 ZeppelinheLm) have a total sugar content of about
50%, with the glucose and galacto3e proportion being
about lsl and, besides the monosaccharide~, uncleaved
lactose also being pre~ent in concentrations of 5-15%.
Such lactose hydroly~ates can be sterilized by auto-
claving without the foxmation of caramelization products
which have an adverse effect on the fermentation of
recombinant yeasts. It has ben found that the gluco~e
present in whey hydrolysates and lactose hydroly5ates
- does not impair in any way the induction o$ FXIIIa
synthesis and the production of FXIIIa by S. cerevisiae
~pMB307~ or S. cere~isiae ~pMB330~. Thi3 wa~ ~n u~expec-
ted finding because gluco e i8 described in the litera-
ture as an inhibitox of the induction of the galacto~e
operon. Furthermore, spray-dried lactosa hy~rolysate can
also be employed, in place of commercial lacto~e hydro-
lysate ~olution~, after it has been ~terilized by
suitable method~, for the fermentation. The high yields
: of FXIIIa described here were achieved with tr~nsfor~ants
which contained either the plasmid pMB307 or p~330, and
thus it can be as3umed khat the hi~h ~ynthesi~ rate~ do
:~ not dapend on particular alleles. It may thus be expected
that a high yield is ensured with this optimized produc-
tion proress even with amino-acid replacements other than
leucine 88 by phenylalanine 88,
:,
` ;' '
?
2031810
- 13 -
FXIIIa concentrations 2 500 mg/l were achleved w~th the
abovementioned medium and ~o-called plasmids pMB307 or
pMB330 on conversion on the l()O-SOO 1 fermen~er scale.
The purified FXIIIa was identical according to all te~ted
criteria with FXIIIa from placentae.
. ,~, ,~ . . , .: ,
~031810
~ 14 --
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16
Fi~ure legend:
Construction of pMB307
The FXIIIa-specific EcoRI/HindIII cDNA fragment was
ligated into the polylinker of pEMBLye~4. Compared with
S the FXIII~ cDNA already described (Amann et al., loc.
cit.) the internal EcoRI Bi~e and ~he 5hine-Dalgarno~like
sequence have been mu ated w:Lthout sltering the amlno-
acid 6equence. These features are repre~ented by ~quare
brackets.
DNA Rep. ~,eans filling in of protruding end~, Lig. means
ligation of the resulting con~truct.
pMB330 differs from pMB307 only by Phe88 ~TTC) in place
of Leu88 (CTC) within the FXIIIa ~equence.
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