JPH01112985A - Base sequence for coding n-terminal amino acid sequence of flagellin having discharge function - Google Patents

Abstract

PURPOSE:To obtain a base sequence capable of coding an N-terminal amino acid sequence having discharge function in flagellin of Escherichia coli or an amino acid sequence having the discharge function equal thereto. CONSTITUTION:The structure of a part (peptide) required to discharge flagellin of Escherichia coli is elucidated by paying attention to a discharge mechanism concerning extracellular production of a desired peptide by integrating a gene capable of coding the desired peptide in a vector and cultivating a host microorganism transformed therewith. Extracellularly discharged hybrid proteins can be readily collected by affinity column chromatography utilizing antiflagellin antibody. Peptides derived from flagellin can be removed from the hybrid proteins to afford the aimed protein.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to DNA encoding a portion of flagellin having an excretion function.

With the recent development of genetic engineering, it is becoming easy to obtain a large amount of the peptide by incorporating a gene that feeds the desired peptide into a vector and introducing it into bacteria such as Escherichia coli. However, since expressed peptides generally accumulate within cells, the produced proteins may inhibit cell growth and proliferation, or if produced in excess, negative feedback may suppress productivity. In addition, in order to collect the target protein, cells must first be collected and destroyed, and the target protein must be purified from the destroyed cells.
This cell destruction product contains many impurities, some of which are harmful to the human body, and it is not always easy to obtain the pure target protein from there.

Proteins constituting cell membranes and secreted proteins are synthesized as precursor polypeptides that have a signal peptide at one end of the amino acid sequence necessary for passing through membranes inside and outside the cell. The signal peptide portion is cleaved and becomes the original protein molecule, which exerts its activity and function.

Purify the peptide of interest accumulated within the cells.

In order to solve this difficulty and improve the productivity of the target protein, attempts have been made to secrete the target peptide to the extracellular space using the above-mentioned biological secretion system. Secretion of Escherichia coli β-lactamase by a secretion vector containing the S. subtilis α-amylase promoter and signal sequence (The Journal
Talented Biochemistry (J, Bioehem,)
95.87-93 (19B4)), secretion of mouse IF11-β by the same vector system (Gene<Gane
) 34.1-8 (1985)), secretory production of human N-α using a secretion vector having an E. coli alkaline phosphatase promoter and a signal sequence.
Journal of Biochemistry (J, Bioc)
het) 97.1429-1436 (1985))
, etc.

In addition to the above-mentioned secretion system, there are also mechanisms by which proteins synthesized within the cell exit the cell.
0 system is known e.g.
Flagellin, which makes up bacterial flagella, is excreted from the bacterial body by this excretion system. Unlike secretion, the synthesized peptide does not have a signal peptide, and is excreted from the bacterial cell as it is without being cleaved by peptidases, thus exerting its function (The Journal of Bacteriology). -(J, Bacterio
l, )159゜1056-1059 (1984))
The gene that feeds E. coli flagellin is called the hag gene, and it has already been cloned into -R322 and -phage (Program and proceedings of the 57th Congress of the Genetic Society of Japan, p. 63 (1985), The Journal.
Bactariology (J, Bactariol)
130.736-745 (1977)), its DN
The entire A sequence has been revealed (The Journal
Talented Bacteriology (J.

Bactriol, ) 16831479-1483
(1986) - The present inventors previously constructed an excretion vector using the hag gene. That is, a linker for inserting an appropriate foreign DNA was inserted into the hag gene at a location that does not damage the structure necessary for excretion. In this case, the peptide encoded by the foreign DNA is excreted from the bacterial cell as a hybrid protein sandwiched between flagellin proteins. When constructing an excretion vector, it is possible not only to use the entire hag gene, but also to delete a part unnecessary for the excretion of flagellin protein and use a part of it, and it is assumed that the unnecessary part is the central part of the hag gene. (Japanese Patent Application No. 61-223484), the essential part of the flagellin protein during L. excretion was still unknown.

Ming tries to decide.

With conventional secretion vectors, the peptides that can be secreted are limited depending on the type of vector. In other words, even if DNA encoding any desired peptide is incorporated into a certain promoter and signal sequence, it will not be secreted;
There are a certain number of peptides that can be secreted, and there are many peptides that are not secreted even if they are incorporated into a secretion vector.

The present inventors discovered that ha which encodes E. coli flagellin.
The g gene was cloned and its entire structure was revealed. Furthermore, we have already shown that the flagellin excretion mechanism can be used for extracellular production of the target foreign protein based on its entire structure (Japanese Patent Application No. 61-223484).
In this case, when constructing the excretion vector, a linker is used to delete the part unnecessary for the excretion of the flagellin protein and insert a DNA part encoding a foreign peptide into that part.
After DNA is inserted, the foreign peptide is finally sandwiched into the flagellin protein and excreted from the bacterial cell as a hybrid protein. It has been suggested that the unnecessary part of the flagellin molecule during excretion is the central part of the molecule, but the part essential for excretion is unknown.

Once the part necessary for flagellin protein excretion has been identified, the target foreign protein can be used for flagellin excretion by linking the DNA that feeds the peptide of that part to the DNA encoding the foreign protein to be excreted. It is possible to eject it using a mechanism.

Furthermore, even if the desired protein is secreted into the medium by a secretion vector, it is still not easy to purify it from the medium, but if it is excreted as a hybrid protein with a partial peptide of the flagellin protein, an anti-flagellin antibody that can be easily prepared can be used. Hybrid proteins can be easily collected from the culture medium using a unique chromatography technique.

As described above, the DNA encoding the essential part for flagellin excretion of the present invention is a very useful gene fragment that enables the construction of an excretion vector.

The present inventors focused on the flagellin excretion mechanism regarding a system for producing the target foreign protein outside the bacterial body.
We have identified the parts (peptides) necessary for flagellin excretion. If the DNA encoding the peptide is inserted into a suitable vector and the DNA encoding the foreign protein to be excreted is linked immediately downstream, the protein will be excreted and accumulated outside the bacterial cell as a hybrid protein with the above peptide. things are predicted. The excreted hybrid operculum can be easily collected by ab initivity chromatography using an anti-flagellin antibody. The desired protein can be obtained by appropriately cleaving the obtained hybrid protein to remove the flagellin-derived peptide. Also, depending on the secretion system, it may be possible to excrete foreign peptides that could not be produced outside the bacterial cell.

Hereinafter, the process of discovering the portion (peptide) necessary for flagellin excretion, which forms the basis of the present invention, will be explained using the hag gene on plasmid pFD201 as an example. In addition, when retesting the present invention, plasmid pBR322/hag
9 (included in FAIKI No. 1232), and -12JAII (included in FAIKEN JOYO NO. 1233), and E. coli in the host.
In some embodiments of the present invention, C600rC600r-:: can be used as host E. coli.
Although the Inl0 strain was used, it is convenient to use the Ni-12JAII strain, which has been deposited with the Microtech Institute, when further testing the present invention. As shown in some of the examples below, the vectors of the invention can also be used in the Ni-12JAII strain without any disadvantages.

Plasmid pFD201 was obtained at the same time as pFD202 described in the example of Japanese Patent Application No. 61-223484, and in the section of Example 2-(C) above, the reaction product of Example 2-(B) was 112 strains of E. coli C600
rC600r-:: Among the transformants that appeared upon transformation into TnlO, it is collected from strains that have a plasmid that can be cut with the restriction enzyme Smal.

That is, pBR322/hag9 obtained in Example ① above
BamH
T4 DNA was digested with I to delete part of the BamHI fragment.
Ligate with ligase to obtain pBR322/hag93.

This is treated with DNaseI, cut, and partially digested with Ba131. This is DNA polymerase! Repair it with Hi
nd m linker was added and ligated with τ4 DNA ligase to obtain pFDl, pFD2 and pFD3. Among them, pFD2 is cut with Hitd■ and partially digested with Ba131. To this, HindM -5ma I -Bgl
II clinker was added and ligated with T4 DNA ligase. This reaction product was introduced into the C600rC600r-: :Inl0 strain, and among the transformants obtained, a strain that exhibited migration properties and whose plasmid was cleaved with SmaI was selected to obtain pFD202. At this time, it was discovered that a strain that did not show migration but had a plasmid that could be cut with 5maI contained pFD201 of the present invention.

The obtained above C600r”m-hag::Inl0 (
From pFD201) to plasmid pFD2 according to the standard method
01 was purified, and the nucleotide sequence in the vicinity of the restriction enzyme SmaI was determined.
The sequence of all mutant hag genes carried by FD201 can be estimated (this can be done in the same manner as the DNA sequence near the SmaI breakpoint on pFD202 described in Example ①-(c) of the specification of Japanese Patent Application No. 61-223484). The base sequence may be determined by the conventional Maxam-Gilbert method or dideoxynucleotide chain termination method.

The entire base sequence of the mutant hag gene carried by pFD201 is as shown in FIG. There is an amino acid translation termination fudon TAA at position 724, and this mutant hag gene is no longer able to synthesize flagellin protein. It can be estimated that a peptide consisting of 241 amino acid residues, consisting of two amino acid residues added to , can be synthesized.

Actually, in the culture solution of JAII (pFD201) strain, in which this pFD201 was introduced into the host E. coli JAIL, the amine terminal side was NH*-Ala-Gin-Val-11@-As.
n-τhr-Asn-5er-Lau-5er-Leu
711g-τhr-Gln-Asn-Asn-I 1e
-Asn-Lys-Asn-Gln-5er-Ala-
It was discovered that Lau-5ar-, a protein with a molecular weight of about 30,000, was accumulated. I will explain the history behind this discovery.

The centrifuged supernatant after culturing JAll (pFD201) was concentrated by membrane filtration, and the resulting sample was separated by conventional 5DS-polyacrylamide electrophoresis, which revealed a large amount of specific pallid matter with a molecular weight of approximately 30,000. It is clearly observed as a band. This protein was extracted from the gel and purified by high-pressure liquid chromatography, and the amino acid residues on the terminal side of the amino groups were determined for the resulting protein preparation. As a result, the sequence of the above amino acid residues was determined.

As described above, flagellin of the JAII (pFD201) strain is synthesized as a peptide consisting of 241 amino acid residues from the amine terminal side and is excreted from the bacterial cell (V).
(see figure).

The present invention is not limited to the base sequence from position 1 to position 726 in the base sequence shown in Figure 1, but the amino acid sequence from position 1 to position 241 shown in Figure 1 or an equivalent excretion function. A nucleotide sequence encoding an amino acid sequence having the following is also within the scope of the present invention.

In particular, AlaSer at positions 240-241 is an amino acid sequence derived from a linker, and it is considered that this is not essential for excretion, so genes that feed the amino acid sequence from positions 1 to 239 are included in the present invention. For the same reason, the base sequence from position 1 to position 717 of the base sequence in Figure 1 is also included in the present invention, and the terminating fudon is also included in the present invention.
Since the base sequence is not limited to A, the nucleotide sequence from position 1 to position 723 is also included in the present invention. Furthermore, since Met at the N-terminus of flagellin is also considered not essential for excretion, the present invention also includes the base sequence obtained by removing the base sequence encoding the N-terminal Met from the above base sequence.

Also, on pFD201, the DN of the step of obtaining the mutant hag gene (see Example ①-(A>) of Japanese Patent Application No. 61-223484)
A cut.

It is known to those skilled in the art that the cleavage and digestion are random, and that it is possible to insert a fudon (DingAA, TGA, or TAG) that terminates the peptide chain at a position that has similar properties but differs in the number of bases and thus the number of amino acid residues. can be easily estimated. Therefore, the present invention is not limited to the nucleotide sequence that hoods the peptide consisting of 241 amino acid residues as described in the Examples, and includes the nucleotide sequence obtained by the above steps as well as the nucleotide sequence having the above properties. hag
All base sequences on genes are included in the present invention.

When retrying the present invention to obtain the nucleotide sequence of the present invention, there is no need to retry the above steps;
The gene can be easily obtained by using FD201) (Feikoken Bibori No. 9671). In addition, E. coli -
12JAIL (9FD201) was released on October 2, 1986.
Since the 2nd, it has been deposited at the Microbiological Technology Research Institute, 1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, as Microbiological Research Institute No. 9671.

K 1 ■Construction and collection of plasmid pFD201 plus, midp
FD201 was obtained at the same time as pFD202 described in the example of patent application No. 61-223484, and pFD
It can be constructed and collected in exactly the same manner as 202. That is, plasmid pFD201 was prepared in Example 2-(C) above by injecting the reaction product of Example 2-(B) into Escherichia coli of 112 strains of C600.
When the r''m-hagj+Tnlo strain was transformed, among the transformants that appeared, the strain was collected from a strain that had a plasmid that was cleaved with the restriction enzyme Sma1.

That is, pBR322/hag9 obtained in Example ① above
(Included in Microtechnology Research Institute No. 1232)
A part of the BamHI fragment is deleted and ligated with τ4 DNA ligase to obtain pBR322/hag93. This is treated with DNasaI to cut it and partially digested with Ba131. This is repaired with DNA polymerase I and Hin
dl[linker was added and ligated with T4 DNA ligase to obtain pFDl, pFD2 and pFD3. Among them, pFD2 is cut with )iitd■ and partially digested with Ba131. To this, HifIdl[[-5mm I-B
gl I [linker was added and ligated with τ4 DNA ligase. This reaction product was converted into C600r-C600r-:TnlO
Among the transformants obtained by introducing the strain into the strain, a strain was selected which exhibited migration ability and whose plasmid was cleaved with SmaI to obtain pFD202. At this time, it was discovered that a strain that did not exhibit migration but was able to compete with a plasmid that was cleaved with SmaI contained pFD201 of the present invention.

■C600rC600r-: +In obtained in Establishment Example of JAll (pFD201)■
1o (pFD201) strain to patent application No. 61-223484.
Of the methods described in Example (1)-(D)-(a),
W3823Hfla-am76 (pBR322/ha
g9) strain C600rC600r-: :TnlO(p
Perform the same operation except replace with FD201) stock,
Prepare pFD201 of cc-DNA.

Next, patent application No. 61-223484 Example (1)-(C
), W3623Hfla-am7
6 strains to JAII strain, and plasmid DNA to pFD2.
The JAII (pFD201) strain was established by performing the same operation except for replacing with 01cc-DNA.

■Culture of JAII (pFD201) JAil (pFD201) strain Wohatato Trifton 1x (
Dayfu Inc.), Yeast Extract O, SX, Sodium Chloride A 0.5%, Agar 1.5%, 7 Picillin 5
LB agar medium containing 0 μg/ml (pH 7, 0-7,
2> and culture at 37°C overnight. Inoculate the resulting flonii into 5ff11 of LB medium (composition omitting 1.5x agar from the above LB agar medium) and culture with shaking overnight at 28°C. Inoculate 1 ml of the culture into 100 ml of the above medium.
Culture with shaking overnight at 8°C.

■Purification of flagellin fragments from culture solution 100 ml of the culture solution of Example
Using A, centrifuge at 5,000 rpm for 10 minutes at 4°C.The obtained supernatant is centrifuged again as described above.As a result, 95ml of supernatant is obtained.Next, this supernatant is filtered through an ultrafilter. (
A filter (UK-10 (43a
+a+φ), Atvanchik Toyosha), inject purified water into the sample chamber of the filter, and add nitrogen gas (1 kg/
Wash the filter with purified water using a pressure of 1.2 cm (cm" pressure). Next, inject 45 ml of culture supernatant into the sample chamber and
Filter and concentrate with nitrogen gas at a pressure of g/c. The sample is 20m1
Once concentrated, 25 ml of the medium supernatant is added, and the above nitrogen gas pressure is applied again to concentrate. When the sample is concentrated to 20 ml, add the remaining 25 ml of medium supernatant and concentrate again. At the point where the sample was concentrated to 20 ml, 50 mM Nac
Add 25 ml of 10 mM sodium phosphate (pH 7,2) buffer containing 1 ml of sodium phosphate and concentrate again. This operation is repeated three more times to finally obtain 20 ml of concentrated sample.

(2) Purification of Flagellin Fragment Example The sample obtained in (2) is purified by 5DS-polyacrylamide electrophoresis using the so-called 1, aemmli method. 5% acrylamide, 0.13% bisacrylamide, 1257 Tris-HCl (pH
6, 8), 0.1% SDS was used, and the separation gel contained 15% acrylamide, 0.4% bisacrylamide, 375
mM Tris-HCI (pH 8,8), 0.1%
SDS was used. As a buffer for aerophoresis, 25mM
1.4 L of τris, 0.192M glycine, 0.1% SDS was used. The electrophoresis tank is a) -5J-10
60SD was used, and the gel thickness was 2 mm.

Sample buffer (125mM
Tris-)1c1 (pH 6,8), 4% SDS,
5% 2-mercaptoethanol, 20% glycerol,
o, o o s% dibromoenol blue 50μm1)
Then, inject it into a sample hole with a width of 4 or 5 mm. Nine sample holes are created per slab (9 lanes), eight for the sample and the remaining one for the molecular weight marker (5DS-P'AGE).
, standard (Low), Bio-Rad) was diluted 50 times with 10mM) Lis-HCl buffer (pH 8,0) containing disodium ethylenediaminetetraacetate (1a+M), and diluted with an equal volume of The sample buffer was added and injected into the 40 μm sample hole. constant current 60
Perform electrophoresis at mA for 3 hours. After electrophoresis, cut out two lanes, one for the sample and one for the molecular weight marker, and add 0.25% Coomassie brilliant blue R-250.
, soaked in 50% methanol and 10% acetic acid for 2 hours and dyed. When electrophoresing the molecular weight marker and electrophoresing the sample after immersing it in 46% methanol and 9% acetic acid was compared, a main band was observed in the sample lane with a molecular weight of around 30,000.

The gel was cut out from the position presumed to occupy the main band from seven unstained sample lanes. This electrophoresis was performed on two slabs to obtain a total of two gel fragments.

Atto's Max Yield Gp protein recovery device AE-35
A dialysis membrane (Spectrum Medical Industries, Inc., Spectra/Pore 6.13) was placed in a 90-inch sample cup.
258G), finely chop the two gel fragments mentioned above, and place them into the gel well of the sample cup. 5D on equipment
Fill with S-polyacrylamide electrophoresis buffer (same as the one in this example section, excluding SDS) and perform electroextraction at 10°C for 2 hours at a constant voltage of 350 volts according to the equipment instructions attached to ATTO. About 2 ml of the solution in the collection well of the sample cup is collected. This recovered solution was centrifugally concentrated using Centrifun-10 (Amicon), and
A sample solution of μm was obtained.

Next, the above sample was subjected to high performance liquid column chromatography ().
It was purified using IPLC). Column Acurepore RP-30, 10 μm (Brown Lee Laboratory)
The flow rate was 1 ml per minute. Of the 320 μm of the above sample solution, 150 μm was charged onto the column, washed with 0.1% trifluoroacetic acid (TFA) for 10 minutes, and then fractionated at 0.5 minutes per fraction tube for the next 10 minutes. 10 of 0.1% TFA
0% → 0%, acetonitrile (CH8CN) O% → 10
Elute with a simultaneous concentration gradient of %G. 12.2 at the 6 minute position
A central peak exhibiting absorption at 20 mμ was detected. Fraction tube 25, 26, 27, 28 Collect the money, 2
ml of purified fraction was obtained.

Next, 50 μm of the above purified fraction was analyzed by 5DS-polyacrylamide electrophoresis using the Laammli method (see Examples in this section). At the same time, flagellin purified from 112 strains of wild-type E. coli (patent application 1986-22) was added to 1 μg of wild-type E. coli.
3484 Example (2) - (B) - (a)) was subjected to electrophoresis, and the intensity of the bands after both staining was measured using a densitometer (Zeinek Softracer.
When compared using a Scan Kong densitometer (Piometh Instruments Inc.), the target protein with a molecular weight of about 30,000 could be detected as a single band, and the purified amount was about 60 μg.

■Example of analysis of amino acid sequence on the terminal side of amine group■
Approximately 15 μg of the sample was transferred to a gas-phase protein sequencer-47.
Using 0A (Applied Biosystems), amino acid sequence analysis of the terminal amino group was performed. The result is NH, -Ala-Gln-Va 1- I 1 e-
As n-Thr-Asn-5er-Leu-5er-
Lau-I 1e-Thr-G 1 n-As n-A
s n-I 1e-As n-1ys-Asn-G 1
n-5er-A] ]a-Leu-X.

This amine terminal amino acid sequence matches the amino acid sequence from the 2nd to 25th amino terminal positions that can be deduced from the DNA sequence of the hag gene (see Figure 1).

If a gene encoding a foreign peptide of interest is connected immediately downstream of the gene of the present invention and inserted into an appropriate expression vector, a fusion protein consisting of flagellin and the peptide of interest can be expressed. , the fusion protein may be excreted by the excretion function of flagellin. Once the fusion protein is excreted, it can be easily purified using an anti-flagellin antibody.

[Brief explanation of the drawing]

FIG. 1 shows the hag gene carried by pFD202 and the amino acid sequence deduced from the gene. roOψ roQI-1roQ-rooh (5<-to aU ψC-啼〇- <-1<'s -oC OO 0 mouth OCS <ψ chestnut<ψ
＜ψ ＜−＜− ＜＜ ＜＜ ＜−aa ○υ F−4＠JE＠a）Q− ←−←−! −4−Qfuchi＜ −＜ −＜ −＜← ←−EIm＋−Q−〇− 〇＜ψ Ro0− Ro＜ψ RoQAri＜＜ m
u<g<<eQ<←←ro −0ko −
←−to O− ＜ψ ←υ uu ＜−＜く ←−
QO of ← (ts Qh Q b C:) 1! <
-a -Ufuchi Q- uo aa <E-4a<←-←-usa
E-1- UJ3 Uυ ←Jl ←-Ro
RO←0 0←Tin ROO> 4 Q a) O←-coE-4ao eqQk
←--QO-0- to QO <+-+ E-1c/) Q< E
-IC/)o:3 ari Oat QO
E-4a E-4t+ <-←-to (,
),,,I C,), -1<<<"-〇&-←ba ←-←h e Qu (jb u-+
ca-ya ←C/) Q<a<
aaW Lo○co LoC Rico 0-υ OO
Heguchi ← - Ro C Koro < ψ Mouth ○ 11: I Ro1-4u ～ku h's <←
M (J-moan- 〇-←-<- u-Ck(+)- a<Q<a< Ql:I CJb-<:) w <-a OU ←C Ua ←'s Q- <-<tna - 〇F-4-〇<hoE-+m cho>pus--QshiheC-'s←υ 啼QbaQ J E-4-(ta <← <-<<<:3:s (ri C- ←C＜-←- Q-CjQ (5〇〇Qfalse Q- ＜-＜ψ 〇- rooOrooku roaO 6o←-璽0roro 〇- 〇-nok-cry ψ O< Ua << em QlJ QO )-1-←-←A a><-←- E-110←-aa at-+ (,)@J <-〇< ←C
/) ua mouth C! 5c! ol+mIs+10〇− ward cQEmIυ ■ku− −←−−kuψtoku−
QO[F]Qfuchi e13Qc c+zE-4c/) m<←
Chest Ori 〇− ←＠J ←υ Ku− ←−Q, J Q(5 Ql:l ←1:IIF−4−＜ψ
<Cut C- <<C< OOQα ←-
←- B<-〇〇@J B0aZ C13<< To E-+ ψ←ψ Ua
So Ori Noku- <ψ Ku- ←- << aa B>Qt)
E-1-〇- ←-ou cl- (x E-1c/) ○Kukuri 0 mouth 01 <-<ψ kuψ ec5Q ro<ku roO< pus〇--←-to←υ clll(,)t ) FIQ-Fl←-←
<T<- loco E-Ih <ko E-4υ o-ku- o, -+ C: Ja oa (jll (,)l-←1 Q-〇, I:I<ψ a<<r-a< RoO:I Roku-RoE+1riψl-u Moaning QA RotoC 啼Q-Riku← (5Q J ←to ←-ku whisper＜ψ＜ψ＜- (,:l-1: a＜＜, -aQt )
QRE-4u ←-<cut ←- <-<<<- ←-←cs E-4rs 啼Qυ ri<ψ ri-Q- ←-<h <1 <-<, -1<< ←-←-ku- a ψ −←
A riQ- << ←-ku← Go to E-4 ←-←- Roku-ro<-〇F-4CC ←-sh < ← Ku(,)&-C:J-<Q ← Qzaa<oa r , rcso &-<<<mouth<ψ roQ, J:l roQ Q ←Q-
←-Q OO <← <-<a<<h<ψ< Q ← a-<h(,)o ← ca <,-+ a OΦQh cp
s << ←a -Q-ro Q(
, ) <F-4E-4+<<. To Q&-a (,) ←＜ψ＜
h U E-4←%J S
"-I:" %-/
% W←-E-4 O←
○ ua<ta f-4u<<←Φ<<<< 00- 〇-1 〇<<0 . 2Q-Ich<ta ui(,) (-)
0Qro<CI:1<<)<< c
p<ca h-0← ← u-a-F-4uc, to<taoa<<:3 ←-a <<<-<aE-4<Qto<< E-I <a o-E-+1 0 ← ○ Mouth Q-Ro<ψ RoE-I To QQ<E-4 Roa
＜-＊＜＜ ← (5E--C0E-1 TOO← ←Q@J
Q, J: I < Q CE-4
<E-4a<a ←ri )-1&-o E-I ku←a<ho
<c, +Q-E71←Q<. Q-Hee Q <E- uJ3 E-4+wou. Mouth<← RoO>RoE-a<-←vTo←h's← ← ←C1:1←-RoΦ-ToQ <<<-acaa<<h a″:3 <Bru ← Hi (5- <-
<*o< aa aa E-1 threat Φto
←u(,)k< ← Qρ S-aU ← ← <E-4<-<no< ← E-4< E-4Q Q ← Q << Q Q Q<<<< ← F-4E-4Q ← Ku ← <<aa<O < ← o c Ku ← uo F-4o f-4s a Q <ova a< C:J ocp. “ ← ← ← ← To Q＜E−4＜＜＜
← <<a<< ← <F-4<<<< Q Q O← 0 <<<a ← OkuE-4<o Hi ← Q <<a<E-@<O< Φ Φ OO < E -40Q ← Ku<E-4E-4QQE-4 <(5'u o o C!1toE-
4<E-4← ← E-40<uou Q ← <E-4< ← o E-4u E-4o< Q F-4← < ← Q ＾ 0 O000* ← To OQ ← Q g<u Q <o E-4 ya ← cpc ← C%, i -a! Fs C)
To O←Q ← 0 0<<<< Q F-4Q Q 00<<E-I< Q O← Q ← Q Kulo Q 0<<<<<<a< u Q << Q Q <<a C:J OC5 トQE-I U*+ Q ← C << ← < ← O Q Q Q Q < Q < C← ← Q Q < ← U << Oo ku ← c Q ← ← Q <aa c C5cc oua @oa Q o<. U＜O＜

Claims (3)

[Claims] (1) Among E. coli flagellin, N has excretion function
A base sequence encoding a terminal amino acid sequence or an amino acid sequence having an excretory function equivalent to the terminal amino acid sequence. (2) The amino acid sequence is Met at the first position or Ala at the second position to the 23rd position of the amino acid sequence shown in FIG.
The base sequence according to claim 1, which is an amino acid sequence up to Gly at position 9 or Ser at position 241. (3) Claim 1, wherein the base sequence is the base sequence from the 1st or 4th position to the 717th, 723rd, or 726th position of the base sequence shown in FIG. Described base sequence.