Method for the extraction of periplasmic proteins from prokaryotic microorganisms in the presence of arginine

The invention relates to a method for the extraction of recombinant periplasmic proteins wherein arginine is used as the extraction agent. In particular, the invention relates to a method for the extraction of a periplasmic protein of interest, which essentially consists in: PA1 (i) suspending the pellet of cells or of cell debris from cells, which cells originate from the culture of a prokaryotic microorganism transformed with an expression vector containing a gene coding for the said protein and means for its expression in the periplasm of the said microorganism, in a buffer solution containing arginine; and PA1 (ii) recovering the protein of interest in the supernatant of the bacterial suspension thereby obtained.

The present invention relates to the extraction of recombinant proteins 
produced by prokaryotic microorganisms, especially by E. coli. 
Increasing use is being made of genetic engineering techniques for the 
production of proteins of interest such as, for example, insulin, 
inter-leukins, growth hormone, and the like. 
Generally, the microorganism is transformed with an expression vector 
containing a gene coding for the protein of interest and means needed for 
its expression such as the regulator signals. The micro-organism is then 
cultured on a suitable culture medium and according to suitable culture 
parameters and, when a sufficient number of microorganism cells has been 
arrived at, the addition of an inducer triggers the so-called expression 
phase, during which the desired protein is produced at high level and 
accumulates. On completion of culturing, the cells in suspension are 
separated from the culture medium, for example by centrifugation or 
microfiltration, and are then subjected to an extraction method which 
frequently begins with an operation of disrupting of the walls of the 
microorganisms. 
The expression of a gene coding for a protein of interest in a prokaryotic 
microorganism can be cytoplasmic, periplasmic or secretory, depending on 
the nature of the means of expression employed with the said gene 
(promoter, terminator, ribosome binding site, signal peptide, and the 
like). 
Cytoplasmic expression enables large amounts of proteins to be obtained. 
However, prior to the extraction of the protein of interest, it is 
necessary, for proteins comprising one or more disulphide bridges, to 
carry out a step of denaturation/renaturation, which represents an 
especially cumbersome and intricate step during production on an 
industrial scale. The denaturation/renaturation step is carried out 
according to traditional means well known to a person skilled in the art, 
using a denaturing agent in the presence of a reducing agent followed by 
renaturation conditions comprising, in particular, a monitoring of the 
redox state of the solution. Among denaturing agents used, most particular 
mention must be made of guanidine hydrochloride, which has been proposed 
in a method for obtaining human interleukin-2. To this end, reference may 
be made, for example, to the document EP-A2-0,145,390. 
With Gram-negative bacteria, little or no use has been made of secretory 
expression systems in which the protein of interest is to be found 
actively in the culture medium, on account of their low productivity. It 
should be noted here that the medium of a bacterial culture at high 
density in a bioreactor is not an ideal residence place for sensitive 
recombinant proteins on account, for example, of the risks of interfacial 
denaturation. 
Periplasmic expression enables recombinant proteins which are, in 
principle, correctly folded to be obtained directly in a space protected 
from the environment and, as a result, represents a judicious choice for 
obtaining proteins, in particular unglycosylated proteins. In this case, 
it is hence not necessary to subject the proteins to a 
denaturation/renaturation step. 
The methods of cell disruption generally used in this field are, for 
example, cell lysis by sonication or by mechanical pressure (French 
Pressure Cell, ball mill), chemical lysis or enzymatic lysis, osmotic 
shock and treatment using chaotropic agents or detergents. These methods 
disrupt the majority of cell membranes, including the plasma membranes and 
membranes of the endoplasmic reticulum, to form a homogeneous suspension 
of cell debris. The nature of the pellet of cell debris which can be 
harvested in general after centrifugation (nuclei, cytoskeleton, 
mitochondria, lysosomes, ribosomes, macromolecules, and the like) is 
dependent especially on the time and the speed of centrifugation (10 
minutes at 1000 g to 3 hours at 150,000 g). 
The difficulties encountered during the extraction operations vary 
according to the type of expression and the extraction methods used, and 
are, in particular: 
loss in yield of the recombinant protein 
loss of biological activity of the recombinant protein 
proteolytic degradation of the recombinant protein 
toxicity of the extraction agents and obligatory monitoring of their 
removal 
difficulty of industrial implementation 
mixing of the periplasmic proteins with cytoplasmic proteins. 
Furthermore, when the proteins of interest produced are hydrophobic or 
charged, they may associate with cell components which are themselves 
hydrophobic or charged, thereby rendering extraction especially difficult. 
Considerable benefit might accrue from undertaking the industrial 
production of recombinant proteins of interest by genetic engineering, but 
this necessitates the development of extraction methods which avoid or 
minimize the above drawbacks. 
In effect, it is not only important to produce large amounts of protein of 
interest, but these proteins must also not be contaminated with the 
extraction agents and must retain their full biological activity. 
Various methods have been proposed for this purpose, especially for the 
extraction/separation of interleukin-2. 
The document EP-A2-0,145,390 describes a method for obtaining 
unglycosylated human interleukin-2 (IL-2) having a specific activity of 
greater than 104 U/mg, which employs a step of separation by column 
chromatography to extract the IL-2. This method also involves a 
denaturation step using guanidine hydrochloride. 
The document EP-A2-0,147,819 proposes a method for obtaining homogeneous 
and pure recombinant interleukin-2. This method consists in culturing a 
microorganism transformed by means of an expression vector containing the 
gene coding for interleukin-2, in causing lysis of the cells, in 
recovering the cell debris, in extracting the IL-2 by washing the cell 
debris with a suitable washing solution and then in purifying the washinng 
solution by chromatography. The washing solutions used can contain a salt 
such as sodium chloride or guanidine hydrochloride, or a detergent such 
as, for example, the product known under the trade name "Triton 
X.RTM.-100". 
According to a preferred variant, the successive use of three washing 
solutions, namely a washing solution containing sodium chloride, a washing 
solution containing a detergent and a washing solution containing 
guanidine hydrochloride, is recommended. 
The document EP-A1-0,337,243 describes a method for purifying human 
interleukin-2 which utilizes a system of two reversed-phase liquid 
chromatography columns. Before the step of purification by chromatography, 
the insoluble fraction of the bacterial cell lysate is extracted with a 
solution containing guanidine hydrochloride to obtain a bacterial extract, 
which is then diluted using a guanidine hydrochloride-free buffer and 
thereafter chromatographed, elution being carried out with an acetonitrile 
gradient. 
It has now been found, surprisingly, that the extraction of a protein of 
interest produced by a prokaryotic microorganism, transformed with an 
expression vector containing a gene coding for the protein of interest and 
means for its expression such as the regulator signals needed for its 
periplasmic expression, may be carried out by suspending the pellet of 
cells or of cell debris from the microorganism, originating from the 
culture of the said microorganism, in a buffer solution, the said solution 
advantageously containing arginine, it being possible for the arginine to 
be in the L and/or D form. 
According to a first aspect, the subject of the invention is the use of 
arginine as an agent for the extraction of periplasmic proteins. 
According to another aspect, the subject of the present invention is a 
method for the extraction of a periplasmic protein of interest, which 
consists in: 
1) suspending the pellet of cells originating from the culture of a 
microorganism, transformed with an expression vector containing a gene 
coding for the said protein and all the regulator signals needed for its 
periplasmic expression, in a buffer solution containing arginine and, 
after a period of contact under appropriate pH, temperature, bacterial 
concentration, and the like, conditions, 
2) recovering the protein of interest in the supernatant of the bacterial 
suspension thereby obtained. 
A variant of the said method for the extraction of a periplasmic protein of 
interest consists in suspending the pellet of cell debris, obtained after 
lysis of the cells originating from the culture, in the buffer solution 
containing arginine. 
Extraction of the periplasmic proteins is especially efficient when the 
extraction buffer consists of an aqueous solution containing arginine at a 
concentration equal to at least 0.4M arginine within the limit of 
solubility of arginine at room temperature in water (in the region of 0.8M 
in pure water and above this in the presence of salts), and when its pH is 
slightly alkaline, preferably equal to 8. 
Arginine is a natural a-amino acid which has been proposed as an auxiliary 
agent for the denaturation/renaturation/substitution of two chains of 
Abbokinase.RTM. (urinary plasminogen activator), in which chains a native 
peptide is partially replaced by a synthetic peptide during this 
operation. To this end, reference may be made to the paper by GA. 
Homandberg and T. Wai in Biochimica et Biophysica Acta, 1990, 1038, 
209-215. 
In the method of the invention or its variant, denaturation/renaturation of 
the protein is not carried out and the arginine participates only in 
respect of the extraction of a protein from a pellet of cells or of cell 
debris from microorganisms. 
Arginine brings about noteworthy effects on the extraction of the protein, 
in respect of both the yield and the biological activity of the protein. 
It was, in effect, found that, for example, the mature form of IL-13 is 
recovered with the method of the invention in yields of greater than 95% 
while retaining the biological activity of the molecule. It should be 
noted that trials of extraction by osmotic shock on the same expression 
system do not lead to comparable yields. 
Comparative trials showed that guanidine.HCl used under the same conditions 
also enables the IL-13 protein to be recovered in a yield of greater than 
95% but, in contrast, the biological activity of the protein thus 
recovered is impaired more than by the arginine method. 
While it is not wished to limit interpretation to some particular theory, 
arginine is thought to act as a mild and biological chaotropic agent, as 
opposed to the powerful chaotropic agents which are denaturing at the high 
concentrations needed, equal to or greater than 5M, in order to effect 
extraction, such as guanidine hydrochloride. 
The method of the invention or its variant may be carried out following any 
method of culture of a microorganism transformed with an expression vector 
containing a gene coding for the protein of interest and means for a 
periplasmic expression of the said protein, such as all the necessary 
regulator signals. 
It is obvious to a person skilled in the art that the method is applicable 
to bacteria closely related to E. coli, that is to say to so-called 
facultative anaerobic Gram-negative bacteria which constitute the 
Enterobacteriaceae group. In this family Enterobacteriaceae, the following 
species are to be found in particular: Escherichia, Salmonella, Erwinia 
and also Shigella, Klebsiella, Serratia, Proteus and Enterobacter. 
Bearing in mind the chaotropic character of arginine, it is also apparent 
that arginine can, depending on the case, advantageously substitute for 
other chaotropic agents. Without it being possible to exemplify on all the 
families of bacteria on account of the diversity of the living systems in 
question, a person skilled in the art will know how to apply and adapt the 
arginine extraction method to his particular case. 
Such culture methods are well known to a person skilled in the art. Methods 
describing the fermenter culture of Gram-negative bacteria are described, 
for example, in Patent EP-360,641 and EP-356,335 reporting the obtaining 
and use of the E. coli strains known as SEBR 1250 and TP 2339. 
When the desired number of cells has been arrived at, the culture is 
subjected to a centrifugation (in general) or a microfiltration, and the 
pellet of biomass obtained is brought into contact with a buffer solution 
containing arginine according to the method of the invention. 
As a general rule, the procedure is performed at a temperature between room 
temperature of approximately 25.degree. C. and 2.degree. C., preferably at 
4.degree. C. 
The contact time of the cell pellet with the buffer solution containing 
arginine must be sufficient to permit passage of the protein of interest 
into the buffer solution. 
In general, when the procedure is performed at 4.degree. C., the contact 
time is advantageously approximately 1 hour. 
The extraction, that is to say passage of the periplasmic protein into the 
medium, continues during the period of contact of the biomass and the 
arginine-containing extraction buffer. The contact time providing for 
complete extraction or an extraction showing no further change in level is 
between 30 minutes and 16 hours. Trials show that satisfactory extraction 
yields may be obtained in the space of a few hours at a temperature of 
4.degree. C. It has also been noted that gentle stirring of the biomass in 
its extraction buffer so as to avoid sedimentation of the pellet of 
microorganisms gives superior results, that is to say higher levels of 
extraction as a function of time. 
The extraction method according to the invention is suitable for extracting 
both hydrophobic proteins such as, for example, interleukins, especially 
IL-13 described in the document EP-A1-0,506,574, and hydrophilic proteins 
such as, for example, growth hormone (hGH). The method of the invention 
simplifies the obtaining of hGH, which normally necessitates the use of an 
osmotic shock for its extraction. 
To carry out the extraction of the protein of interest directly on the 
suspension of the cell pellet, a buffer solution containing arginine at a 
concentration of between 0.4M and 0.8M will be preferred. 
When it is desired to carry out the extraction of the periplasmic protein 
of interest on the pellet of cell debris according to the variant of the 
method of the invention, the procedure is the same as is used in the 
method of the invention up to the step of obtaining the cell pellet 
obtained after centrifugation or micro-filtration, and disruption of the 
cells is then performed according to methods well known to a person 
skilled in the art. Methods of cell disruption are described, for example, 
in C. T. Choma and H. Yamazaki, Can. J. Microbiol., 1981, 27, 547-550; L. 
O. Ingram, Journal of Bacteriology, 1981, 146, 1, 331-336; N. G. Nossal 
and L. A. Heppel, Journal of Biological Chemistry, 1966, 241, 13, 
3065-3072; R. Bennett, D. R. Taylor and A. Hurst, Biochem. Biophys. Acta, 
18(3), 512-521 (1966), and in the collective work Fermentation and enzyme 
technology, Chap. 12, 239-309, J. Wiley and Sons publishers (1979). 
The pellet of cell debris harvested, as a general rule, after 
centrifugation is resuspended and then brought into contact with a buffer 
solution containing arginine. The contact time of the suspension of cell 
debris with the buffer solution containing arginine must be sufficient to 
permit passage of the protein of interest into the buffer solution. In 
general, for a temperature of 4.degree. C., the contact time providing for 
almost complete extraction is 48 hours. Similarly, it was noted that 
gentle stirring of the biomass in its extraction buffer, thereby avoiding 
sedimentation of the cell debris, gives higher levels of extraction as a 
function of time. 
This variant of the extraction method according to the invention is 
suitable for extracting especially periplasmic proteins of interest which 
are strongly associated with the cell membranes, such as, for example, 
interleukins. 
It is well known to a person skilled in the art that the extraction buffer 
containing arginine according to the invention may also contain an 
auxiliary detergent which will have the effect of improving the yield 
and/or the rate of extraction of the protein of interest. Among auxiliary 
detergents which may be used, a person skilled in the art will be able to 
choose from those which enable the advantages of using arginine as 
extraction agent, especially the retention of the biological activity of 
the protein of interest, to be preserved. Among these mild auxiliary 
detergents, there may be mentioned, for example, alkyl glycosides such as 
alkyl maltosides, nonyl .alpha.- or .beta.-D-glycopyranosides, octyl 
.alpha.- or .beta.-D-glycopyranosides or alkylcarbamoylmethyl .alpha.- or 
.beta.-D-glycopyranosides such as, for example, Hecameg.RTM., the very low 
toxicity of which suggests the possibility of allowing it to appear in 
trace amounts as formulation agent in the final product. 
To carry out the extraction of the protein of interest from the suspension 
of the pellet of cell debris, it will be preferable to use a buffer 
solution containing arginine at a concentration of between 0.4M and 2.5M, 
it being possible for a concentration of 2.5M arginine to be obtained 
especially in the presence of salts. 
Moreover, it was found that arginine exerts a considerable beneficial 
effect on the yields of secreted recombinant periplasmic protein if it is 
added at unfamiliar concentrations much higher than those encountered in 
the culture media manufactured from commercial protein hydrolysates, and 
which enable the arginine requirements of the strain employed to be 
covered. 
Furthermore, it was found that the beneficial effect exerted by arginine is 
especially considerable if the arginine concentrations added to the 
culture medium are between 2 g/l and 10 g/l. 
Thus, according to another aspect, the subject of the present invention is 
a method for the culture of a prokaryotic microorganism transformed by 
means of an expression vector containing a gene coding for a protein of 
interest, which consists in culturing the said microorganism in the 
presence of arginine at a concentration equal to at least 2 g/l, and 
especially at a concentration of between 2 g/l and 10 g/l. 
A person skilled in the art will optimize this arginine concentration for 
each particular case. 
This method is especially suitable for the production of proteins having 
activity of the cytokine type, especially IL-13, as described in the 
document EP-A1-0,506,574.

The invention will now be described in greater detail by means of the 
EXAMPLES below, given only by way of illustration. 
EXAMPLE 1 
Extraction of periplasmic IL-13 from E. coli in the presence of arginine on 
cell pellet. 
1/ Flask culture: 
In this example, E. coli strain RB 791 (Roger Brent, PNAS 78 (1981) pp. 
4204-4208), transformed with the plasmid p922 obtained according to 
methods similar to those defined in Patents EP 360,641 and 356,335 and 
whose DNA sequence is the sequence SEQ ID NO:1, was used. 
The different sequences which constitute this plasmid p922 are shown below. 
PROMOTER SEQUENCE (SEQ ID NO:2) 
The hexanucleotides TTGCTT and TATAAT characteristic of the promoters in E. 
coli are shown in bold characters 
##STR1## 
SEQUENCE OF THE UNTRANSLATED 5-PRIME REGION OF THE MESSENGER (SEQ ID NO:3) 
The ribosome binding site is shown in bold characters. The sequence CAT 
located at the 3-prime end of this sequence is a portion of the 
hexanucleotide recognized by the restriction enzyme Nde I 
##STR2## 
SEQUENCE CODING FOR THE IL-13 PRECURSOR (SEQ ID NO:4) 
The sequence in italics corresponds to the sequence of mature IL-13. The 
sequence which is not in bold characters is a linker sequence linking the 
end of the sequence coding for IL-13 to the hexanucleotide recognized by 
the restriction enzyme BamH I 
##STR3## 
This strain E. coli RB 791/p922 was set up in preculture overnight at 
30.degree. C. with stirring at 200 rpm on L medium (Luria broth described 
in Molecular Cloning, A Laboratory Manual Sambrook, Fritsch, Maniatis; 
Cold Spring Harbor Laboratory Press, 2nd edition 1989) containing 100 mg/l 
of ampicillin. From this preculture, a further flask of L medium was 
inoculated such that the initial OD (OD=optical density at 600 nm, OD=1 
corresponds to 400-450 mg biomass/liter) was 0.6. After waiting for one 
hour, the culture was induced with 1 mM IPTG (isopropyl 
.beta.-D-1-thiogalactopyranoside) and culturing was continued for 3 hours. 
The samples of the bacterial suspension were centrifuged and the bacterial 
pellets thus recovered were suspended in the extraction buffers below, 
such that the final OD was 10, this being equivalent to 4.5 g 
biomass/liter, at the time of extraction. 
The extraction buffers used in this example are the following: 
A: 0.8M arginine pH 8.0 corrected with HCl in Milli-Q.RTM. water 
(Millipore) 
B: 5M guanidine.HCl in Milli-Q.RTM. water without pH correction. 
Extraction was performed in 1 hour at 4.degree. C. with gentle magnetic 
stirring. 
To measure the efficiency of extraction, samples equivalent to 1 ml of 
culture suspension with an OD of 0.2 were removed, and the corresponding 
bacterial pellets obtained by centrifugation at 5,000 g for 10 min were 
applied to 16.5% polyacrylamide gel after denaturation with SDS. The 
bacterial suspensions were also centrifuged and their supernatants were 
desalted by ultrafiltration (Millipore Ultrafree-MC filtration device with 
a cut-off threshold of 5,000 Da) before being applied to gel. The gel 
itself was visualized by Western blotting using an anti-CHO IL-13 antibody 
and quantified with a PhosphorImager.RTM. (Molecular Dynamics). The 
anti-CHO (Chinese hamster ovary) IL-13 antibody used in this example was 
obtained by immunizing rabbits. 
It was found in this example that extraction in the presence of 
guanidine.HCl or alternatively in the presence of arginine is virtually 
complete for the mature form, with extraction yields greater than 99% in 
both cases. It was also noted that, in the supernatant extracted in the 
presence of arginine, the precursor form of IL-13 is not seen, in 
distinction to the extract obtained in the presence of guanidine.HCl. 
2/ Fermenter culture: 
E. coli strain RB 791/p922 was set up on L medium with 100 mg/l ampicillin 
and incubated at 30.degree. C. with stirring to constitute a preculture. A 
100 ml volume of this preculture was used as inoculum for an MBR brand 
fermenter of total volume 2.5 liters. Culturing was performed in a volume 
of 1.2 liters on a medium whose composition is given below and under the 
conditions defined below. 
Medium for fermenter--E. coli strain RB 791/p922 
The formula is given for 1 liter final, the volume of the inoculum is to be 
subtracted. 
1. Dissolve in 700 ml of Milli-O.RTM. water: 
______________________________________ 
Component Mass/liter 
______________________________________ 
EDTA 1 g 
FeSO.sub.4.7H.sub.2 O 
45 mg 
MgSO.sub.4.7H.sub.2 O 
1.5 g 
K.sub.2 SO.sub.4 
0.75 g 
CaCl.sub.2.2H.sub.2 O 
32 mg 
NaCl 1.45 g 
KCl 5 g 
HY-SOY .RTM. 75 g 
L-methionine 1.4 g 
Tryptophan 1 g 
Trace elements* 2 ml 
Yeast extract 10 g 
______________________________________ 
Make to 800 ml with Milli-Q.RTM. water, autoclave 30 min at 120.degree. C. 
2. Filter through 0.2 .mu.m in 100 ml of Milli-Q.RTM. water: 
______________________________________ 
Glycerol 
15 g 
K.sub.2 HPO.sub.4 
7.1 g 
______________________________________ 
The glycerol concentration will be maintained at between 10 and 15 g/l 
during culture. 
3. At the time of induction add: 
______________________________________ 
IPTG 1 g 
6-Aminocaproic 0.65 g 
acid 
HY-SOY .RTM. 40 g 
L-cysteine 0.3 g 
______________________________________ 
The volume of this addition is not included in the other calculations. 
Solution of trace elements 
This is used in the proportion of 1 ml/liter. 
For 1 liter of Milli-Q.RTM. water final, dissolve in 800 ml: 
______________________________________ 
mass/1 
______________________________________ 
H.sub.3 BO.sub.3 
3 mg 
NaMoO.sub.4.2H.sub.2 O 
4.8 mg 
MnSO.sub.4.H.sub.2 O 
59 mg 
CoCl.sub.2.6H.sub.2 O 
23.8 mg 
CuSO.sub.4.5H.sub.2 O 
8.7 mg 
ZnSO.sub.4.7H.sub.2 O 
13 mg 
AlCl.sub.3.6H.sub.2 O 
60 mg 
KCr(SO.sub.4).sub.2.12H.sub.2 O 
6 mg 
KI (added at the 
60 mg 
time of use) 
NiSO.sub.4.6H.sub.2 O 
2.6 mg 
______________________________________ 
Add 100 ml of concentrated HCl. Make to 1000 ml with Milli-Q.RTM. water. 
When the OD has reached 58, the expression of IL-13 is triggered by the 
addition of IPTG at a concentration of 1 g/l and continued for 5 hours. 
The fermenter culture parameters were as follows: 
pH=7.4 
T=30.degree. C. 
pO.sub.2 =40 mm Hg regulated by stirring, with a flow rate of air of 
between 1 and 3 liters/min. 
The methods of extraction and of measurement of the biological activity 
which are applied are the same as those described in section 1 above. 
It is found that extraction--on a bacterial pellet obtained in a fermenter, 
no longer in a flask--in the presence of guanidine.HCl or alternatively in 
the presence of arginine is virtually complete for the mature form of 
IL-13, with extraction yields of greater than 97% in both cases. 
EXAMPLE 2 
Biological activity of the IL-13 thus extracted 
The extracts obtained in the presence of guanidine.HCl or of arginine in 
Example 1 were desalted by ultrafiltration as described above. After 
serial dilution, they were brought into contact with an IL-13-dependent 
subclone of the B9 cell line. The IL-13 activity of the diluted samples 
induces the growth of B9 cells, and the half-proliferation concentration 
was determined. Cell growth was stopped after 3 days of contact by adding 
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and 
measured in a spectrophotometer by the absorption of the blue colouration 
produced at 565 nm. The IL-13 biological activity was expressed in ng/ml 
relative to an IL-13 standard which was itself calibrated against the 
candidate international standard, obtained from an CHO IL-13 culture, 
obtained by immunizing rabbits according to N. Vita, Archives of 
Biochemistry and Biophysics, 1983, 225, 2, 436-445. 
The results obtained appear in TABLE II below. 
TABLE II 
______________________________________ 
Biological 
Specific 
Trial on B9 
IL-13 in activity in 
biological 
cell line ng/ml ng/ml activity 
______________________________________ 
Control 500 500 100 
Arginine 3,200 1,376 43 
Guanidine 4,300 1,098 25 
______________________________________ 
The above results show that the specific biological activity of the 
arginine extract is, before any other subsequent purification operation, 
greater than that of the guanidine hydrochloride extract. 
EXAMPLE 3 
Extraction of periplasmic hGH from E. coli in the presence of arginine on 
cell pellet 
The strain SEBR 1250 (EP-360,641 and EP-356,335) was set up in preculture 
overnight at 37.degree. C. with stirring at 200 rpm on L medium (Luria 
broth) containing 100 mg/l of ampicillin. From this preculture, a further 
flask of L medium was inoculated such that the initial OD was 0.2. After 
waiting for one hour, the culture was induced with 1 mM IPTG and culturing 
was continued for 3 hours. The samples of the bacterial suspension were 
centrifuged, and the bacterial pellets thus recovered were suspended in 
the extraction buffers such that the final OD was 10, this being 
equivalent to .about.4.5 g biomass/liter, at the time of extraction. 
The extraction conditions were as follows: 
______________________________________ 
Chaotropic agent 
pH T time 
______________________________________ 
0.8 M Arginine 
8.0 22.degree. C. 
20 hours 
0.8 M Arginine 
8.0 4.degree. C. 
20 hours 
______________________________________ 
To measure the efficacy of extraction, samples equivalent to 1 ml of 
culture suspension with an OD of 0.2 were removed, and the corresponding 
bacterial pellets obtained by centrifugation at 5,000 g for 10 minutes 
were applied to 16.5% polyacrylamide gel after denaturation with SDS. The 
bacterial suspensions were also centrifuged and their supernatants applied 
to gel. The gel itself was visualized by Western blotting using an 
anti-hGH antibody, and quantified with a PhosphorImager.RTM. (Molecular 
Dynamics). The anti-hGH antibodies used were obtained by immunizing 
rabbits. 
Analysis of the bands obtained with the PhosphorImager.RTM. enables the 
conclusion to be drawn that the extraction of human periplasmic hGH 
produced in E. coli in the presence of arginine is efficient. In this 
example, a yield of at least 60% may be achieved in the presence of 0.8M 
arginine, pH 8.0, T 22.degree. C. and a period of 20 hours, and an 
extraction yield of greater than 80% may be achieved in the presence of 
0.8M arginine, pH 8.0, T 4.degree. C. and a period of 20 hours. 
Since hGH is a hydrophilic protein, it may be concluded from this that 
recombinant proteins differing greatly in nature, accumulated in the 
periplasm of E. coli, may be extracted simply in the presence of arginine. 
EXAMPLE 4 
Extraction of periplasmic IL-13 from E. coli on cell debris in the presence 
of arginine 
1/ Fermenter culture 
In this example, E. coli strain TP2339 (EP 360,641 and EP 356,335), 
transformed with plasmid p922 obtained according to methods similar to 
those defined in EXAMPLE 1 was used. 
E. coli strain TP2339/p922 was set up on L medium with 100 mg/l ampicillin 
and incubated at 30.degree. C. with stirring to constitute a preculture. A 
100 ml volume of this preculture was used as inoculum for an MBR.RTM. 
brand fermenter of total volume 2.5 liters. Culturing was performed in a 
volume of 1.2 liters in a medium and under conditions defined below. 
Medium for fermenter--E. coli strain TP2339/p922 
Calculated for a final volume of 1.2 liters, the culture medium consists of 
the addition of one liter of autoclaved phase and 0.1 liter of filtered 
phase whose compositions are described below, and of 0.1 liter of 
preculture defined above. 
1/ Autoclaved phase (1000 ml): 
Dissolve in 900 ml of Milli-Q.RTM. water: 
______________________________________ 
Mass/1 
______________________________________ 
Tricine 360 mg 
FeSO.sub.4.7H.sub.2 O 
280 mg 
CaCl.sub.2.2H.sub.2 O 
6.7 mg 
MgCl.sub.2.6H.sub.2 O 
1.27 g 
K.sub.2 SO.sub.4 
8.71 g 
NaCl 500 mg 
KCl 5 g 
Hy-Case (SF) .RTM. 
25 g 
Yeast extract 18 g 
Trace elements* 1 ml 
L-arginine 1.5 g 
______________________________________ 
Adjust the pH to 7.4 with KOH solution and then make to 1000 ml with 
Milli-Q.RTM. water. Autoclave 30 minutes at 120.degree. C. 
2/ Filtered phase (100 ml) 
Filter under sterile conditions through a 0.2 .mu.m membrane: 
______________________________________ 
Glucose 
20 g 
Glycerol 
50 g 
K.sub.2 HPO.sub.4 
5 g 
______________________________________ 
The glucose concentration will be maintained during culturing at a 
concentration of between 5 and 15 g/l. 
When the OD has reached 40 (approximately 16 g of dry matter/liter), the 
expression of IL-13 is triggered by the addition of IPTG at a 
concentration of 1 g/l and continued for 5 hours. The culture parameters 
were as follows: 
pH=7.4 regulated with 3N HCl and KOH 
T=37.degree. C. 
pO.sub.2 =50 mbar regulated by stirring, with a flow rate of air of between 
1 and 3 liters/min. 
2/ Recovery and grinding of the bacterial bodies 
One liter of culture suspension is centrifuged for 20 minutes at 
.about.6400 g. The pellet is taken up in the same volume of 10 mM Tris 
buffer, 1 mM EDTA, 1 mg/l pepstatin, pH 8, with mechanical stirring using 
a propeller-type paddle. 
Grinding is accomplished in a Manton-Gaulin press at a pressure of 700 bars 
in two runs. The ground preparation as it is may be stored at -80.degree. 
C. in this example. 
3/ Extraction 
After thawing, 5 ml of the ground preparation with an OD equal to 75 (30 g 
of dry matter/liter) are removed and then centrifuged for 50 minutes at 
23,300 g. 
The pellet thereby obtained is taken up in one third of the initial volume 
with 0.1 mM Tris buffer, pH 7.0, and then made to the initial volume with 
a solution containing arginine such that the final arginine concentration 
is 2.5M and the pH 8.0. 
For this example, an auxiliary detergent (Hecameg.RTM. at a final 
concentration of 20 g/l) was combined with the arginine. 
The suspension of cell debris made up in this way is placed at 4.degree. C. 
on a rotary stirrer at 300 rpm for 2 days. 
The suspension is then centrifuged a final time for 50 minutes at 23,300 g, 
the supernatant constituting the expected extract. 
4/ Biochemical analysis and analysis of biological activity 
a) Assay of total proteins was performed by the Biorad.RTM. "Protein Assay" 
method. 
b) The method of assay of recombinant IL-13 is that used in Example 1. 
Yield of IL-13 thus extracted: the results obtained are described in the 
following table: 
______________________________________ 
In the suspension 
In the 
of cell debris 
supernatant after 
before extraction 
extraction 
______________________________________ 
Total proteins 
324 .mu.g/ml 
108 .mu.g/ml 
Recombinant IL-13 
575 ng/ml 390 ng/ml 
______________________________________ 
It was found in this example that the extraction carried out on cell debris 
in the presence of 2.5M arginine and an auxiliary detergent enabled an 
extraction yield of approximately 70% to be obtained. 
EXAMPLE 5 
Expression of IL-13 in the presence of arginine in the culture medium 
E. coli strain RB 791/p922 was cultured on L medium with 100 mg/l 
ampicillin in the presence of different concentrations of arginine. 
Induction was triggered 1 hour after inoculation by the addition of 1 mM 
IPTG, and culturing was continued for 3 hours. 
The samples of bacterial pellets--equivalent to 1 ml of culture suspension 
with an OD of 0.2--and the corresponding samples of supernatant were 
applied to gel, visualized and quantified as described above. The results 
are given in the table below: 
______________________________________ 
Sample OD end of culture 
IL-13 in ng/l OD 1 
______________________________________ 
Control 1.17 388 
Arginine 2 g/l 
1.24 455 
Arginine 4 g/l 
1.24 600 
Arginine 8 g/l 
1 720 
______________________________________ 
It is apparent that, under the experimental conditions and in the 
expression system in question: 
arginine increases the expression of periplasmic IL-13 from 2 g/l, and 
substantially from 4 g/l, 
growth of the bacterium is slowed down at a concentration of 8 g/l 
at these concentrations, arginine does not cause leakage of IL-13 into the 
supernatant. 
The value of the arginine extraction method according to the invention is 
the ability to use protein extracts as they are or with a minimum of 
treatment in tests of biological activity. 
This simplification of the extraction method affords an advantage both for 
the industrial production of recombinant periplasmic proteins, and for 
screening by assaying the biological activity on the laboratory scale in 
relation, for example, to mutated proteins. 
In distinction to guanidine.HCl, frequently used as extraction agent, 
arginine does not attack the materials employed in industry, in particular 
steels. Furthermore, arginine is a non-polluting agent, which thus does 
not require an expensive effluent treatment process. 
The value of expressing a periplasmic protein in the presence of arginine 
at concentrations equal to at least 2 g/l, and especially at 
concentrations of between 2 g/l and 10 g/l, in the culture medium is 
demonstrated by the increase in the yield of secreted recombinant protein 
obtained in vivo. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4410 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
TCGAGTGGGTTTGAGGCGATCACACTTCTGTTAACGCAGAACCTAAACGCATCTCGACTG60 
CACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCA120 
GGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTT180 
TTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTTCGAGCTGA240 
CTGACTGTTGCTTATATTACATCGATAGCGTATAATGTGTGGAATTGTGAGCGGATAACA300 
ATTTCACACAGTTTTTCGCGAAGAAGGAGATATACATATGAAAAAGATCCTGGCGTTAGC360 
TGCGCTGACTACCGTTGTATTCTCTGCGTCCGCCTTCGCTGGCCCTGTGCCTCCCAGTAC420 
TGCCCTCAGGGAGCTCATTGAGGAGCTGGTCAACATCACCCAGAACCAGAAGGCTCCGCT480 
CTGCAATGGCAGCATGGTATGGAGCATCAACCTGACAGCTGGCATGTACTGTGCAGCCCT540 
GGAATCCCTGATCAACGTGTCAGGCTGCAGTGCCATCGAGAAGACCCAGAGGATGCTGAG600 
CGGATTCTGCCCGCACAAGGTCTCAGCTGGGCAGTTTTCCAGCTTGCATGTCCGAGACAC660 
CAAAATCGAGGTGGCCCAGTTTGTAAAGGACCTGCTCTTACATTTAAAGAAACTTTTTCG720 
CGAGGGACGGTTCAACTGAAACTTCGAAAGCATCATTATTTGGGATCCGGCTGCTAACAA780 
AGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCT840 
TGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATG900 
TACCAAGCTTGGCCGGATCAAAGTTTTGTCGTCTTTCCAGACGTTAGTAAATGAATTTTC960 
TGTATGAGGTTTTGCTAAACAACTTTCAACAGTTTCAGCGGAGTGAGAATAGAAAGGAAC1020 
AACTAAAGGAATTGCGAATAATAATTTTTTCACGTTGAAAATCTCCAAAAAAAAAGGCTC1080 
CAAAAGGAGCCTTTAATTGTATCGGTTTATCAGCTTGCTTTCGAGGTGAATTTCTTAAAC1140 
AGCTTGATACCGATAGTTGCGCCGACAATGACAACAACCATCGCCCACGCATAACCGATA1200 
TATTCGGTCGCTGAGGCTTGCAGGGAGTCAAAGGCCGCTTTTGCGGGATCGATCCGCGGA1260 
AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG1320 
CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC1380 
CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAG1440 
TGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCG1500 
GTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGCTGGTGGTTAACGGCGGGAT1560 
ATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCG1620 
CAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAG1680 
CATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACAT1740 
GGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTT1800 
ATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGC1860 
GATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATG1920 
GGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAAC1980 
ATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGAT2040 
CAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCC2100 
GCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAAT2160 
CGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAG2220 
CAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGC2280 
CATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCAC2340 
GCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGG2400 
TTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAA2460 
GGTTTTGCGCCATTCGATCTACGCCGGACGCATCGTGGCCGCAAACCAACCCTTGGCAGA2520 
ACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCCGCGTTG2580 
CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGT2640 
CAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC2700 
CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCT2760 
TCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC2820 
GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA2880 
TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA2940 
GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG3000 
TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG3060 
CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT3120 
AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA3180 
GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG3240 
ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA3300 
AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA3360 
ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC3420 
CCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATG3480 
ATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA3540 
AGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGT3600 
TGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT3660 
GCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC3720 
CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC3780 
GGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA3840 
GCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG3900 
TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG3960 
TCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA4020 
CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA4080 
CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA4140 
GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA4200 
ATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG4260 
AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT4320 
CCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA4380 
AATAGGCGTATCACGAGGCCCTTTCGTCCC4410 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 282 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
TCGAGTGGGTTTGAGGCGATCACACTTCTGTTAACGCAGAACCTAAACGCATCTCGACTG60 
CACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCA120 
GGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTT180 
TTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTTCGAGCTGA240 
CTGACTGTTGCTTATATTACATCGATAGCGTATAATGTGTGG282 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
AATTGTGAGCGGATAACAATTTCACACAGTTTTTCGCGAAGAAGGAGATATACAT55 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 425 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
ATGAAAAAGATCCTGGCGTTAGCTGCGCTGACTACCGTTGTATTCTCTGCGTCCGCCTTC60 
GCTGGCCCTGTGCCTCCCAGTACTGCCCTCAGGGAGCTCATTGAGGAGCTGGTCAACATC120 
ACCCAGAACCAGAAGGCTCCGCTCTGCAATGGCAGCATGGTATGGAGCATCAACCTGACA180 
GCTGGCATGTACTGTGCAGCCCTGGAATCCCTGATCAACGTGTCAGGCTGCAGTGCCATC240 
GAGAAGACCCAGAGGATGCTGAGCGGATTCTGCCCGCACAAGGTCTCAGCTGGGCAGTTT300 
TCCAGCTTGCATGTCCGAGACACCAAAATCGAGGTGGCCCAGTTTGTAAAGGACCTGCTC360 
TTACATTTAAAGAAACTTTTTCGCGAGGGACGGTTCAACTGAAACTTCGAAAGCATCATT420 
ATTTG425 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
GGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCA50 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 200 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
CCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTT60 
TGCTGAAAGGAGGAACTATATCCGGATGTACCAAGCTTGGCCGGATCAAAGTTTTGTCGT120 
CTTTCCAGACGTTAGTAAATGAATTTTCTGTATGAGGTTTTGCTAAACAACTTTCAACAG180 
TTTCAGCGGAGTGAGAATAG200 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 241 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
AAAGGAACAACTAAAGGAATTGCGAATAATAATTTTTTCACGTTGAAAATCTCCAAAAAA60 
AAAGGCTCCAAAAGGAGCCTTTAATTGTATCGGTTTATCAGCTTGCTTTCGAGGTGAATT120 
TCTTAAACAGCTTGATACCGATAGTTGCGCCGACAATGACAACAACCATCGCCCACGCAT180 
AACCGATATATTCGGTCGCTGAGGCTTGCAGGGAGTCAAAGGCCGCTTTTGCGGGATCGA240 
T241 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1252 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
CCGCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT60 
TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATG120 
AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTT180 
TCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCA240 
GCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGCTGGTGGTTAACG300 
GCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCAC360 
CAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG420 
CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAAC480 
CGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGA540 
GATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTA600 
ACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGT660 
CTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACG720 
CCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGT780 
TAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTT840 
CGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAG900 
ATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGC960 
CAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCA1020 
GCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGT1080 
TCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACG1140 
TTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATAC1200 
CGCGAAAGGTTTTGCGCCATTCGATCTACGCCGGACGCATCGTGGCCGCAAA1252 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1905 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
CCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCA60 
TCTCGGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA120 
AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTT180 
CCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG240 
TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTC300 
AGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC360 
GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA420 
TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT480 
ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATC540 
TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAA600 
CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA660 
AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA720 
AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTT780 
TTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC840 
AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC900 
ATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC960 
CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATA1020 
AACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC1080 
CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGC1140 
AACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA1200 
TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA1260 
GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA1320 
CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT1380 
TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGT1440 
TGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG1500 
CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA1560 
TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC1620 
AGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG1680 
ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG1740 
GGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG1800 
GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATG1860 
ACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCCC1905 
__________________________________________________________________________