JPS6091987A - Incidence plasmid indicating excessive production of specific protein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides proteins such as interleukin-2, β-interferon and β-2 tamase in Escherichia coli (g,
The present invention relates to inducible expression cilasmids for overproduction in E. coli.

Advances in recombinant DNA technology have made it possible to isolate specific genes or portions thereof from higher organisms such as humans and other mammals, and to transfer those genes or fragments to microbial species such as bacteria or yeast. I made it. The transferred gene is replicated and propagated as the transformed microorganism self-replicates. As a result, the transformed microorganism may have the ability to produce any protein that the gene or fragment coats, whether it is an enzyme, hormone, antigen, antibody, or any other protein.

Such scientific technology has already been applied to matostatin (Itakura).
et al., 5science Vol. 198, No. 1056-1066
(1977)), component A and B chains of human insulin (Goeade et al. Proc. Natl.
Δc+ad.

Soi, USA Vol. 76, pp. 106-110 (197
9), Human Growth Hormone (Goedde 1 et al., Nature Vol. 281, pp. 544-548 (19
Useful polypolymers such as (see GB 2,068,970 A) and human interferon (see GB 2,068,970 A) have been used in the modification of bacteria to produce peptide products. U.S. Patent No. 4,322,499 Nos. 2-7
The column summarizes some of the techniques used in DNA recombination technology.

Human interleukin-2 (L-2), a glycoprotein with a molecular weight of about 15,000 daltons, has also been produced by recombinant DNA technology. Tan JguchL et al., Nature, Vol. 602, pp. 305-310 (198
In 3rd year), cDNA encoding engineering L-2
The cloning and expression of it in cultured monkey cells to produce biologically active peptides has been described. Mr. F'u, jita and others are Pr.
0c, Natl, Acat5. , Elci, l.
l5A Vol. 80, pp. 7437-7441 (1983)
describes the sequence of the engineering L-2 gene.

Mr. Dθvoθ and others are Nualsic Ac1cls Re
5earch No. 5earch 07-4623 pages (198
3), and Roθenberg et al. 5c.
ience Vol. 223, pp. 1412-1415 (198
4), the IL-2 gene was expressed under the control of the trp promoter in Escherichia coli to produce biologically active IL-2.
It is described that the two products are obtained at a concentration of about 5 to 10 times the total protein.

Many recombinant DNA techniques use two types of compounds: transfer (lids) and restriction enzymes.

A transfer vector is, inter alia, a DNA molecule containing genetic information that ensures its own replication when transferred to a host microbial strain. Examples of transfer vectors commonly used in bacterial genetics are plasmids and the DNA of certain bacteriophages. "Plasmid" is the term used for autonomously replicating DNA units found in microbial cells other than the host cell's own genome.

"Restriction enzyme" is a term used for hydrolytic enzymes that can catalyze the cleavage of DNA molecules. Many of these enzymes are DN
It will recognize specific short sequences in A and, depending on the particular enzyme and the sequence of DNA it recognizes, will cleave or catalyze the cleavage of the DNA into precise, reasonably short fragments. Heterologous after cleavage
A gene or gene fragment can be inserted into a plasmid by end-joining techniques at the cleavage point or with rearranged ends adjacent to the cleavage point. The term "heterologous" refers to genes not normally found in a particular microorganism, such as E. coli, or polypeptide sequences not normally produced by that microorganism. [The term homologous J refers to a gene or polypeptide sequence commonly found in or produced by a particular microorganism.

If the gene is inserted properly for the portion of the plasmid that directs the transcription and translation of the encoded DNA message, the resulting vehicle can be used for the expression of the protein encoded by the inserted gene. DNA
The term "expression" in recombinant technology refers to the operation in which the protein encoded by the transferred gene is actually synthesized by the transformed bacterium.

The manner in which gene expression is regulated in E. coli is well understood. Genes that code for proteins that perform related functions in cells tend to cluster together in continuous sequences. This group is called ``Oha Ron''. All genes in the operon start at the codon that codes for the N-terminal amino acid of the first protein in the sequence and are transcribed in the same direction continuing until the O-terminus of the last protein in the operon. Ru.

At the beginning of the operon closest to the N-terminal amino acid codon are sequences for the promoter-operator and liposome binding site (5hinθ-Dalgarno sequence).
There are regions of DNA called control regions that contain various control elements including. The function of these sites is to cause these genes under their control to be expressed in response to the needs of the bacteria. The control region functions that must be present for expression to occur are transcription into metsenger RNA complementary to the DNA template and its transcription into metsenger R.
This is the translation of NA into proteins on organelles called liposomes.

Expression of the first gene of the sequence is initiated by transcription and translation at the position encoding the N-terminal amino acid of the first protein of the operon. The expression of each gene along the flow from that point is also assembled in such a way that the signals respond to at least a different set of environmental cues, such as its own control region and a stop signal or other signals in sequence until it encounters a stop signal or other signals. will be started. Although there are many variations in the details of this general scheme, the important fact is that in order to be expressed in prokaryotes such as E. coli, genes must be linked to control regions with transcriptional and translational functions. This means that it must be properly positioned. Even small changes in the promoter sequence can increase the probability that RNA polymerase will bind to the promoter and increase the rate of transcription. Mutations in the receptor region or regulatory genes can prevent repressor binding and thereby greatly increase transcription.

If recombinant DNA technology is to fully maintain its promise, systems should be devised that can optimize the expression of gene inserts and thus ensure that the intended protein is available in high yields. be. The commonly used I-2 catamases and lactose promoter-operator systems have not fully utilized the capabilities of this technology from a yield standpoint. A more efficient promoter system is the tricyto7an (trp) promoter system, which has been used to enhance the expression of foreign genes in bacteria. For example, Yorotsuno ξ Patent Application No. 36776 describes an attenuation body (attenua).
Plasmid expression of human growth hormone using the trp promoter-otor system in which the tor ) region has been deleted has been described.

HalleWe11 et al. Gene Vol. 9 No. 27-47
(1980), the E. coli trp promoter, ~t
Plasmid pBR322 suitable for the expression of foreign genes containing the rpE gene and about 15-trpD genes.
Derivatives of are described. Fidman et al., Nature, vol. 291, pp. 503-506 (1981
describe the preparation of recombinant plasmids that direct the synthesis of hepatitis B nuclear antigen and β-lactamase/hepatitis B surface antigen fusion hoIJ peptide under the control of the trp operon regulatory region.

Ru5set et al., Gene Vol. 17, pp. 9-18 (1
(982), the trp pro-single-o-transferase 4l-tp (base pair) Alul restriction fragment from the later region was p.
Cloned into the Pvull breakpoint of BR322, Pv
The preparation of plasmids carrying several novel trp-promoter promoters by regenerating the ull breakpoint is described.

Ohristie et al. J, Mo1. Biol, Vol. 143, pp. 335-341 (1980) describes plasmid pLD102. Guarente
Oele, Vol. 20, pp. 543-553 (1980)
The plasmid pLG400 is described in 2010).

European Patent Application No. 52002 describes a plasmid which has an insertion site for a DNA fragment adjacent to the trp promoter whose attenuator has been removed. Yorotsu 7mi Patent Application No. 48970 specifies t
The preparation of mature human fibroblast interferon using a plasmid containing the rp promoter system is described. European Patent Application No. 67540 describes chemically inducible promoter-operator fragments, such as lac, and fragments of second promoters with greater signal strength, such as trp.
, a hybrid promoter containing
is listed.

GB Patent Application No. 2,104,901A describes portable promoters such as lac and trp for expressing foreign genes in E. coli.

The use of is described. lac o is exemplified by the use of lon.

The present invention relates to novel inducible plasmids useful for the overproduction of proteins of a large number of homologous and heterologous structures in bacteria. A typical bacterium in which cilasmids can be used is Escherichia coli, and typical proteins that can be produced in excess include interleukin-2, β-interferon,
Includes N-lactamase and β-lactamase. The present invention also relates to novel promoter-operator fragments, microorganisms transformed with the plasmids of the invention, methods for producing these plasmids, methods for incorporating them into microorganisms, and methods for overproducing the encoded proteins. "Biologically pure culture" as used to describe the microorganisms of the present invention means a genetically homogeneous culture.

The novel promoter-operator fragment of the present invention is the GO in the -35 region of the (+) fragment (5'→3').
OGA UGGOT, and (11) the first eight base pairs (5'→3') are O.
GC! GC! ATC (an approximately 17 base pair sequence between the -35 and -1o regions of the fragment, which is the normal OG (!GTAG)).

Plasmids of the invention include those containing promoter fragments described herein having sequences (1) and (11). These also have pK()
P43, pBRWIL-2, pK()PI3-19R
-1; r'p6, pKGP36・trp and pTrp
HF: Includes Engineering L-2.

Typical plasmids of the present invention are as follows.

pKGP36 See Fig. 4A pK, GP43 See Fig. 3 pAP16 See Fig. 5 pAPF engineering F9 See Fig. 6 pKGP12-2 See Fig. 7 pKGP13-19 See Fig. 7 pKGP9-25 See Fig. 8 pKGP13-19R-trp6 See Figure 30 pKG
P13-19R See Figure 9 pKGP9-25R See Figure 8 pKGP12-2R See Figure 7 pBRK Engineering L-2 See Figure 14 pKGP36-trp See Figure 15 pTr pl)! ! Engineering L-2 See Figure 15 The present invention also encompasses such plasmids that have mutated somewhat over time.

The method for preparing plasmids pKGP36 and pKGP 43 consists of recombining fragments of known plasmids of pLDIQ2 and pBR322 types by the following method: (1) ];
and Ba1l, (If) trp-promoter-operator-containing H
The pal/Bal fragment was isolated and purified.

(Ill) I(pal/Ba11 fragment was modified by limited digestion with Taq I, and (V) the modified fragment was transformed into c1a of pBR filter 22.
l Insert at the cutting point.

From the reaction mixture, pP43 and pKGP36 containing trpP,O, containing the novel promoter-o-retar fragment of the present invention were isolated.

The method for preparing plasmid pAP16 of the present invention consists of the following steps: (1) Restricting the pLG400 type plasmid using Petl and inhibitor [l to obtain P , q t l/Hi
ndIII fragment was obtained, (tl) the plasmid of type pKGP36 was restricted with Psti and Hl ndlll and the fragment containing the promoter-operator was isolated and purified, and (lii) the promoter-operator-containing fragment of pKGP36 was obtained. pLG400P
Bt)/H1nall fragment is ligated.

The method for preparing plasmid pApn engineering F9 of the present invention is as follows (1)
) Transfect the engineered β cDNA fragment with HaeIL/'Hi
ncll to obtain the HaθJl/H1nall fragment, and this fragment was isolated and purified.
ll/H1nall fragment was obtained, and this fragment was isolated and purified, (tii) the plasmid of type pAP16 was
ll and (Iφ) Step (10 fragments are ligated with the fragments of step (lii).

Plasmids of the invention pKGP 12-2 and pKIl
) The method for preparing P13-19 consists of the following steps: (1) Converting pAP16 type plasmid into Hlndl
(11) Restrict the fragment from step (+) with Petl to obtain a blunt cut fragment by filling the cut point with the Klenow fragment of E. coli DNA polymerase, and (11) restrict the fragment from step (+) with Petl to
Amine terminus and pK of lactamase gene (AMP)
σP36 tryptophan promoter is rate-8
hine-Da'1garno (P, O, -8, D,
): a Pstl fragment containing the s-nit was obtained and this fragment was purified, (iii) the pAPF engineered type F9 plasmid was restricted with Petl and the carboxyl terminus of the β-lactamase gene and the IFN-βcD
The fragment containing the carboxyl terminus of NA was purified and the (lφ engineered βQ DNA fragment was edited to a base pair 140
ATG of the blunt end with /i) the s t l fragment and (v) the fragments of steps (tl), θ11) and 4v) are ligated.

The method for preparing plasmid pKGP9-25 of the present invention consists of the following steps: (1) pAP16 type plasmid is transformed into alnaBl 'f
restriction using C and E. coli D at the H1ndll breakpoint.
Fill in the Klenow fragment of NA polymerase to obtain a blunt cut fragment; (11) Digest the fragment from step (1) with BamH.

(ii) Restrict the plasmid of pAPF type F9 with Hpal to obtain three fragments; Fragments were obtained that were randomly disrupted and terminated at the BamHl breakpoint.

(v) Process (
11) into the pAP16 fragment.

Plasmid of the present invention pKGP 12-2R% pKGP
13-19R and I) The preparation method for KGP9-25H consists of the following steps: (1) Restriction using pBR322 type plasmid gold lasmid I and BamHi to obtain the Petl/Bam old fragment; (it) 1p) pxGpiz-z type, (b) pKG
Plasmids of type P1319 or (c) pKGP9-25 were restricted with Pstl and the amine terminus of the β-lactamase, pKGP36 P,O,-8,D, unit and the amino terminus of the edited engineered FN-β gene. Purify the fragment containing.

OiD P3847 type plasmid was transformed into Pstl and B
glI and purified the fragment containing the carboxyl-terminal fragment of the engineered FN-β gene and
Steps (1), (iiD and Step (II) (7)
(a), (b) i fc C. Connect the fragments of (a).

Cilasmid of the present invention pKGP13-19R-trI)6
The preparation method consists of the following steps. (1) A plasmid of type pKGP43 was restricted using Hpai and l1lind and the Hlnal [l end and Hp
A fragment with a l end was obtained, and (II) pKGPL s-19R type plasmid was
The Hpal fragment is obtained by restriction using pal, and (Ijl) the fragment of step (1>) and the fragment of step (11) are ligated.

The method for preparing plasmid pBRE engineering L-2 of the present invention consists of the following steps: (+) plH40 type plasmid is restricted with Pstl to obtain a cDNA fragment containing the gene for engineering L-2 (11 ) The fragment of step (1) is treated to form a blunt 3'-end starting at the OOT codon; ligate the entire adapter molecule encompassing the r codon and containing the H1ndV breakpoint; (Iv) Step OiD fragments are combined with H1n411 and Bt
(■) The pBR322-type plasmid was restricted with Hlndl [and Pvul, and the large DNA fragment was isolated, and the fragment containing the L-2 gene of Step (HlndVPvul of ψ [@Connect to piece.

The method for preparing cilasmid pKGP36/trp of the present invention consists of the following steps: (1) pKG, P36 type cilasmid is restricted with Hpal and Pstl and a fragment containing the tetR gene is purified; (Ii) pKGPl3 -19R-trp-6 type cilasmid restricted with Hpa 1 and Potl and t
Purify the fragment containing rpP, O, and ligate the fragments from steps (1) and (11).

The method for preparing plasmid pTrpK L-2 of the present invention consists of the following steps: (1) pBRE engineering, L-2 type plasmid is transformed into Hlna
(II) Restrict the plasmid of pKGP36+"trpW with Hlndll and Pstl and purify the fragment containing the engineered L-2 gene;
Purify the fragment containing rp P, O, f, and then ligate the fragments of step (1) and step (11).

The method for modifying a microorganism that incorporates the novel plasmid of the present invention consists of the following steps: (1) growing bacterial cells to a specified optical density, and (It) treating with a tm calcium solution. In particular, the cell wall is modified (11D competent cells are mixed with a ligated DNA mixture containing the new cilasmid, and the mixture is cultured to allow plasmid insertion).

Analysis of the modified microorganisms containing the now cloned cilasmids is accomplished by culturing the cultured cells of step 0ψ on the selected medium and then assaying.

The method of producing excess protein from modified bacterial cells of the present invention consists of the following steps: (1) growing the modified bacterial cells in an enriched medium to an optical density of at least 2 at 6 DO nm; □ Grow in the ground, (11) dilute the grown cells 1:10 in minimal medium, and (11) dilute the diluted cells with β-indole acrylic acid (
β-Engineering AA).

The medium containing diluted cells and β-enzyme AA contains interleukin-2 and /17+21. can be analyzed for overproduced proteins by assaying for activity associated with the protein of interest, such as β-2 tamase and FN-β activity. Alternatively, the produced protein can be labeled with radioactive amino acids and the radiolabeled protein can be separated and analyzed.

The plasmids shown in the figures are not to scale. Base pair distances were measured clockwise.

Figure 1 shows the vector pAOYo 177s and the tryptophan-deleted t that fuses the tryptophan 177 s and tryptophan 177 s and tryptophan 177 s and tryptophan 177 s and tryptophan 177 s that fuses the C gene of tryptophan to the tryptophan leader liposome binding site.
Conventional plasmid p containing the rpΔLD102 sequence
LD102 is shown.

Figure 2 shows the conventional plasmid pBR322.

Figure 3 shows P, 0. Figure 3 shows the restriction endonuclease map of the novel plasmid pKGP43 containing the β-2 lactamase gene with the orientation being that of the β-lactamase gene.

FIG. 4A shows P, 0. is nato2cycline resistance (tθt
R) Restriction endonuclease map of the novel plasmid pKGP36 containing the β-2 taxamase gene with gene orientation.

Figure 4B shows the novel P,0. fragment pair t
A comparison of rp P, O, fragments is shown.

Figure 5 shows pKG fused to the β-galactonase gene.
A novel plasmid pA encompassing p, o from P36
A restriction endonuclease map of P16 is shown.

Figure 6 shows the new plasmid pAPFF9 and its digestion formula.

Figure 7 shows the new plasmids pKGF12-2 and pK
Restriction endonuclease map of GP13-19 and pAP16 Petl blunt snip fragment, pAPFIF
9 shows their preparation using a three-way ligation of the 1FN-β fragment of the 9Pstl fragment and the edited I'etl blunt end.

Figure 8 shows an alternative m4J method for the preparation of plasmid pK()P9-25 using pAPF engineering F9 and Ba13i exonuclease and ligating into pAP16.

Figure 9 shows the old Pstl/Ban fragment from pBR322.
pstl fragment from IIKGP13-19 and pst
Figure 1 shows the preparation of a new plasmid p'KGP13-19R that trilaterally ligates the FN-β fragments.

Figure 10 shows the new cilasmid pK()PI3-19R-t
The digestion, ligation and transformation steps for forming rp6 are shown.

FIG. 11 shows the cDNA sequence of the IFN-β gene from plasmid p3847.

FIG. 12 shows the plasmid pKGP12-2R of the present invention.

The DNA sequence of the transcription unit containing the P, 0°-B, D, fragments characterizing pKGP 13-19R and pKGP9-25R is shown.

Figure 13 shows a fragment of approximately 400 base pairs containing the Hpal breakpoint from pLD102.

Figure 14 shows the edited L-2 gene fragment from plH40, which is obtained by restriction of pBR322.
1Wl by ligating to the ll1 n dIIL/'Pvu I fragment.

-2 vector preparation is shown.

Figure 15 shows pKGP36-trp and pTrpHi-L-2 and pKGP36-trp from Pg.
tI/H1ndnl trp p, o.

The pBRI fragment contains the L-2 gene and part of the ampicillin gene! ! The latter pTrpM! by ligation to a fragment from pTrpM! (2) Preparation of L-2 is shown.

Although expression of foreign genes in E. coli is generally enhanced by the presence of an effective iomomo such as the tryptophan fromomotor, plasmids expressing genes under the control of this promoter can be unstable. Foreign gene cloning, editing, and manipulation can be difficult due to deletions and rearrangements. One of the features of the present invention is that it provides a "deficient" promoter-oleator that can be used to clone and edit foreign genes in a very stable manner and that can then be converted into an effective promoter after the genetic manipulation is complete. It's about doing. An advantage of such systems is long-term retention of the sequences required for expression of foreign genes.

Plasmids and Microorganisms A number of the novel plasmids of the present invention are described with respect to their restriction endonuclease maps and DNA sequences shown in the figures. One of the most distinctive regions of some of these plasmids is the promoter-or-lator and Sh
This is a fragment that includes the ine-Dalgarno sequence.

A preferred embodiment of the present invention is the plasmid p shown in FIG.
It is KGP43. The Hpa l cut point located between the Ola l and H1 nall cut points is P, O, -8, D
, notes the presence and orientation of fragments (indicated by arrows). In this new plasmid, the β-2 catamase gene is tryptophan p, o. -8,D.

Directing downward from the fragment. 5hinθ-Lla1ga
The rnO sequence was determined by 5hinθ et al.
54, pages 34-38 (1975).

This map includes the known dimensions and restriction map of pBR322 (see Figure 2), tryptophan P.0.0 from pLD10'2 (see Figure 1). -8. D. It is constructed based on the known dimensions and restriction maps of the fragments, as well as independent restriction mapping and DNA sequence analysis of the hybrid plasmid pKGP43.

The molecular weight of pKGP43 is 2.8 x 10' Daltons (approximately 4
700bp). HpalL/'raq l fragment (
Tryptophan P, O, -E1. D, containing fragment) indicates the absence of tryptophan in the plasmid or β
- Provides the ability to code for overproduction of β-lactamase in the presence of engineered AA or both.

Said P, 00-8. D. The base sequences in the included fragments do not change the stability of the hybrid plasmid.

The following base pair distances further characterize plasmid pKGP43: Pstl-EcoRI, 750
bp:F! coRI-Hpal, 100bp: Hpa
l-Hlndll, 300bp; Hlndll-B
amH7, 345 bp. This distance was determined by agarose and polyacrylamide gel electrophoresis and is an approximation. The gcoRl and Pstl single restriction sites can be used for insertion and expression of foreign genes.

Another preferred embodiment of the invention is plasmid pKGP36, shown in Figure 4A. Hpal cutting point i P
, O,-8,D, is located in the array and facing the direction. This new plasmid has a molecular weight of approximately 50×11)'
Dalton, and the tetR gene is A, 08-8. D.
Pointing downward from the fragment. Restriction fragment size and placement are determined as described for pK()P43.

The presence of the p,o・,-8,D fragment indicates that the plasmid has H
Provides the ability to express foreign genes cloned into ln'dIII or BamHI restriction sites at low levels. After cloning and genetic manipulation, pKGP3
6 P. 0. is reconstituted in a single one-step cloning to form a valid trp P,O.

The resulting plasmid can then direct high-yield synthesis of the cloned protein. We describe this plasmid by the following base pair distances, i.e. retl-vc
oRl, 750bp; zcoRl-clal, 23
bp: ('1aI-Hpal, 565'bp', 0
1al-01al+60Dbp: Hpal-01ai
, 35bp; Hpal-Hlnall, 59bpS
Inhibition nail-BamHl.

It is 345bp. The promoter region of pKGP36 was further characterized by sequence analysis.

Plasmid pKGPsb contains a novel P,0. Contains the sequence.

This new P,0. is the −10 region of trp P, O, and tr via the Taq l breakpoint during cloning.
It consists of a novel 135 region fused to the p-10 sequence.

See Figure 4B. This new sequence consists of a new sequence of 6 base pairs starting with the T2A base pair and ending with the A:T base pair.
Contains the region (5'→3'). These two base pairs frame the entire 135 region of the trp7p mouth motor. However, these 2 in pKGP36
The four base pairs between the two base pairs differ from those in the trp promoter. The distance between the -35 and -10 regions of pKGP36 is 17 base pairs, the same distance as in the trp i lomotor. This new promoter has the basic structure to function as a promoter, but is less efficient than the trp promoter. This may be advantageous if the ultimate goal is to fuse a foreign gene such as F'N-β to an effective promoter such as the trp or 1aO promoter.

Another preferred embodiment of the invention is the plasmid pAP16 shown in FIG. Molecular weight approximately 4.8x1
06 dalton plasmid vector pAP16 is p
It is a general purpose vector useful for fusing foreign genes to the P, Oo-〇, D, sequence from KGP 36.

β-galactosidase gene 1d used, ATG
It has no initiation codon and cannot direct the expression of β-calactoctase alone. The β-galactosidase gene from pL()400 is described as P from GP36. 0. -8. D
, fused to regulatory elements to form pAP16.

This buffer produces undetectable levels of β-galactosidase. However, functional amine-terminated gene fragments include P, O, -8, D, regulatory elements and β-
When inserted in frame with the galactosidase gene, a fusion protein exhibiting β-galactosidase activity can be produced. This indicates that the P of pKGP36 is 0. greatly facilitates the selection of foreign gene fusions to.

Plasmid pAP16 is Pst l/H of pKGP36
1ndl [l P, O.

Containing fragment and Pst I/H1 of plasmid pLG400
Fusion between the ndnlΔ'laa engineering 2-containing fragment and the main 2-containing fragment. This plasmid can be further characterized by the restriction map below, namely Pet(-Hlndlff
, 1300bp; O:Lal-01a! ,600bp
: Hpal-Hlndlll, 39bp; H tile 1-
BamHl, 45 bp. Plasmid pAP16
In order to include the △1aO engineering 2 sequence fused to the promoter-osulator sequence of plasmid pKGP36 to fuse the foreign gene into the p, o, and leader 〇, D, sequences and in frame with the Δlac engineering Z gene. Create a unique vector. Expression from the promoter-operator can be detected by a typical β-galactosidase assay, e.g. pAP? 1
F'9 (see Figure 6) occurs. The resulting plasmid is then used to engineer the sequence between the tryptophan leaders S, D, and the foreign gene of interest.

This plasmid can also be used to study the expression of heterologous structural genes in E. coli.

Another use of this plasmid is for the antigenic determinant Δla
This is a fusion to the c engineering 2 gene. This would allow the synthesis of fusion antigens for diagnostics or vaccines.

Plasmid pAP 16 can be used for the cloning and manipulation of any foreign gene sequence that carries all of its own ATG initiation codon when fused in frame with the Δ1hcIZ gene. Plasmid pAPi6 is an E. coli tac
P, 0. and provides an alternative method for using aaC bacterial strains. Since the expression of β-galactosidase activity from pAP16 # conductors carrying fusion proteins such as pAPF engineering F9 is under the control of the tryptophan operator, plasmid-directed β-galactosidase activity remains above background in any E. coli host. can be measured.

The tac P,O, fusion system requires a specific strain containing the entire deletion of the chromosomal β-galactosidase gene. This is necessary because the same inducer (lactose and PTG) induces the expression of both the plasmid and chromosomal β-galactosidase genes.

Plasmid activity is therefore indistinguishable from chromosomal activity. In pAP16, plasmid-directed expression of β-galactosidase activity can be induced by l) depletion of tryptophan gold from the medium or addition of β-galactosidase.

These inducing conditions permit expression of plasmid-directed β-galactosidase activity without causing chromosomal β-galactosidase expression. Strains lacking β-galactosidase, such as LG90, give very low efficiencies (5-10%) for correctly configured molecules. f)
Strains containing the l/l: tac Z gene, namely MM 294 and HB 101, yielded correctly assembled molecules with efficiencies of 90 and 95%, respectively, when transformed with the same ligation mixture.

Another embodiment of the invention is plasmid pAPFIF9, shown in FIG. This plasmid with a molecular weight of approximately 5.1 x 10" daltons is Ps t I-Hpa
I.

1320 bp; Hpal-Hindll [, 39 bp
;Hlndllll-Pstl, 207bp;Petl-
Hlndlll, 270 bp; and for H1nd-B
It has a restriction map of amT-11,6bp. In this plasmid, the IP”N-β amino terminus is pKGP
36 P, O, -8, D.

fused between the regulatory element and the β-galactosidase gene.

Restriction mapping, DNA sequence analysis, and bioassays demonstrate that fusion proteins with β-galactosidase activity are
- used to indicate that it is directed by the β initiation codon, ATG K.

P, O, -B, D, control element and engineering FN-βAT
Manipulation of the DNA between the G codon and the G codon can be performed to minimize production of fusion proteins as monitored by β-galactosidase assays. - If proper expression of the fusion protein is obtained from the pKGP 36 P, O, -S, D sequence, the reconstruction of the authentic FN-β gene will result in the aac engineering 2 sequence containing the entire engineered FN-β carboxyl-terminal gene. can be obtained by substituting the fragment (see Figure 7). Tryptophan P, 0. can be reconstituted from pKGP36 p,o to efficiently express FN-β or other foreign genes. Other preferred embodiments include pKGP 13-19R.
and pKGP 13-19R-trp 6.

Plasmid pKGP 12-2.13-19 and 9-
25 is about 5. lX1 (16 dalton molecular length) and Pst 1-Hpal 11320 bp; Hp'a
l-06al, 27 bp; O6a'l-'P
stl, 145 bp; Pstl-Hlndlll
, 270bp and Hlndlll-EFLmlll,
6 bp, eru limit map f) 'F! j be marked.

Plasmid pKGP 12-2R, 16-19R:I?
and 9-25R is a molecular chain of approximately 3.4 x 106 daltons and Pstl-Hpal, 13301) p; Hpal
-Otal, 27 bp and 04al-Pstl,
It has a restriction map of 145 bp. Plasmid pKGP
13-19R-trp 6 has a molecular weight of approximately 3.7X10'
Daldor jip and Pet-Hpal, 1530
bp;Hpal-C1al e27bp> and 04a
l-Pstl, with a restriction map of 145 bp.

Plasmid pB, REIL-'2, has the characteristics of 1, molecular weight approximately 1.7 x 106 Daltons, and HlMlil-IL-2,5tu containing the 几-2 gene.
l breakpoint, 450 bp% xL-2stu1 breakpoint without IL-2 gene - H1ndlll 2325
It has a restriction map of bp.

Plasmid pKG, P36・trp has a molecular weight of approximately 3.0
X10' Dalton and Hlndlll-PstJ 3
586 bp%Pstl-cm1゜775 bp; Tr
p P, O, f inclusive T le (Jlal -C4a I
, 790bp; C'tal-Hlndllll 4
It has a restriction map of bp.

Plasmid pTrp+EIL-2fi molecule approx. 2. lX
HL containing 106 Daltons and IL-2 gene
ndllll -pstl, 2υ00 bp and 'rr
p l', U, i including Pstl-) (1ndll
It has a restriction map of 1569 pages.

Microorganisms suitable for transformation include Escherichia coli (Kscheri-
Chia coli) hf R1 bacteria are included. Examples of suitable strains include Fi, Ni-12 strain, HB 101 strain, and MM.
294 strain and R1 (including 1 strain) K-12 strain is preferred.

Methods As will be appreciated by those skilled in the art, the editing, rearrangement, and other manipulations described herein are performed between the control elements and the first ATG codon that initiates translation of the desired protein. These manipulations are performed at the amine end (front part) of the gene. Easily Assayable Proteins Rearrangement events of interest can be easily assayed when the negative total coating-T gene is fused to the amine terminus of the outer rice gene. For the purposes of the present invention, the β-galactosidase gene and the tryptophan quaternary Ron control element from E. coli are selected. The level of enzyme activity is directly proportional to the number of β-galactosidase molecules present. Thus, by fusion to the amine terminus of the β-galactosidase gene, such as the engineered FN-β gene, the levels of the hybrid molecule expressed in E. coli can be determined by mjFLl colorimetric analysis.

Plasmids pKGP 43 and pKGP 36 were obtained by the following procedure. That is, after digestion with the restriction enzymes Hpa■ and JJatI, blasmi F
' pLD 102 to E. yJ tributophane promoter-o-transmitter, Shine-f) algarn
o I took out the DNAJ coin. No9? A piece of DNA containing the desired DNA was prepared and modified with the restriction enzyme Taql.The obtained modified piece was transformed into plasmid pBf?
322 was subcloned into the 01a1 restriction site. The construction procedure described above resulted in two expression plasmids containing sequences from the citrp operon in opposite orientations.

Plasmid pKGP 431't can direct the expression of genes subcloned into the ElcoRl and Pstl restriction sites or into any site within the β-lactamer heptagene. Cilasmid pKGP36 is a novel hybrid triwotophan P,0. and)]
During the in4 restriction region, fc can direct low-level expression of genes subcloned into any site within the offspring.

The novel plasmid of the present invention can be used to overproduce heterologous and homologous polypeptides such as interleukin-2, β, etc. when the appropriate genes are inserted into the plasmid. - Preferred use of these plasmids for overproduction of lactamase and human fibroblast interferon.Plasmid pKGP 43
for the production of β-lactamase, plasmid pKG
Particularly preferred is the use of p13-19R-trp 6 for the production of FN-β and the use of pTrpB L-2 for the production of interleukin-2. Is β-lactamase M-compatible as a screening agent for drug research and as a drug for removing residual nijirin from nIl liquid samples in analytical preparations? have Interferon is a natural molecule produced by one cell in response to viral infection. This molecule makes them resistant to viral infection when exposed to #4+1. Interleukin-2 and 11 may influence cell-mediated immune responses in mammals. Linf is talented. For example, it functions as a regulator of T-lymphocytes.

The accession numbers of the deposited plasmids of the present invention are as follows.

Plasmid ATCO deposit number p, KGP 36 39413 pKGP 43 39414 pAP 16 39415 pKGP 13-19R59416 pKGP 13-19fltrp6 39412pTr
pEIL-2,39750 Examples are provided below to illustrate the present invention. Unless otherwise specified, all parts and fractions are by weight and all temperatures are in degrees Celsius.

Example 1 Plasmid pLD 102k host E. coli W6110t
Culture 1 of the rp055 strain was grown in a standard clarified t7' bath and subjected to an isostatic centrifugation procedure.

2 μg of this plasmid was injected into
It was digested with Batl and separated on a 5% acrylamide gel. Several fragments were observed that moved around the 400 base pair (bp) range. When the Hpall/Bat] fragment is further digested with Hpal, fragment 1 disappears, which means that Hpan/Ba/! , trp in one fragment
P. O, -El, D, fully indicates the presence of the sequence. This fragment was then purified and subjected to limited digestion with Taq I to determine whether the expected Taq I restriction site was present. T
aq] Polyacrylamide gel analysis of the digested proboscis showed the existence of the expected break point.

400b with Hpa l cutting point (Fig. 16)
The p fragment was partially digested with Taq f as follows. i.e. 250.0 μg'i of fragments, final volume 1
0 μ in 1 unit of Taq Ik at 65°C.
Digested for 2 minutes. Ota l plasmid pBR622
was used to dissipate it into dead money. 1100n of vector DNA i final 8R101ttK ligase buffer, adenosine triphosphate (ATP) 500M and T4 DNA
Partial T of 4 Cl O'bp in 2 units of A ligase
Combined with 4μt of aql digest. Above mixture sound 12
The cells were cultured at ℃ for 48 hours.

One 12 strains of E. coli used for transformation were 294 (en
dA-, 11θdR-, tJll-, pro''') and HEiυ1 (prO-4eu-thl-1taCy
-1h6dR1endA-2rθCA-1rpe L2
L), ara 141gatK2. xyj-5, mtt
-1+θupE44). tIII transformed
The cells were grown in LB medium with optical density (o, D,) in 20 d of culture solution.
550 until it reached 0.5. The cells were separated by 5U in a separator (Sorvallss-54) rotor.
The cells were centrifuged for 6 minutes at OOrpm to form pellets, which were resuspended in K 51J mM G11LOj210 and incubated on ice for 1 hour. Next is Gai! One cell was made into a let shape again and 422 ml of 50 mM 0aO
I had a IB again during V. Next, competent thin M8
! , and incubated for an additional 30 minutes on ice.

These are used for direct transformation or 0aO6
2i@Liquid overnight storage 1-24:00 ri-i] and then used. 10 μt of the above ligation mixture? Combiten)
7k1 (ILItl 200μ) was added into the medium, and the mixture was incubated in the dark at 67°C for 5 minutes 1'.
11-cell DNA mix V (LB Bai Jya (0.8ml)
f was added, and this was then cultured at 37°C for 11 hours. A portion (100 μt) of ampicillin was added to 25 μg/mt.
was applied onto LJ3 plates containing . plus i1-
``DNA tri Birnboim and Do 17
The sea bream was prepared using the method described in Nucleic Acids Ros, Vol. 7, pp. 1516-1526 (1979). The plasmid containing the tryptophan promoter promoter and Shine-Dalgarno sequences is designated 'ipK()P.

Plasmid DNA was purified by an elongative method (C!lewell) and from 4400 bp to 495 Cl bp
The dimensions ranged from . 1 Hpa I restriction site”?
Plasmids with inserts of 100 bp and 7' were subjected to restriction enzyme analysis to determine the orientation of the tryptophan promoter fragment and its position with respect to the unique restriction sites of pBR322. 1 and the orientation of the fragments were determined by agarose and polyacrylamide gel electrophoresis of restriction enzyme digests. Vector pKGP 43.44.45
and pKc+P 66 were selected for further studies.

The expression vector pKGP 43 contains tryptophan P oriented such that the β-lactamase gene is in its control.
, O,-S,D, has fragments. This was inferred from the restriction map in Figure 3. The efficacy of the comb6 current vector was demonstrated by analyzing the expression of β-lactamer 7. i 4th
p] (G
P 43.44 and 45 (pKGP 44 and 45
pKGP 43 (an independent isolation of the same construct as pKGP 43) was grown in LB medium with shaking to approximately 5 x 10 cells/-. β-indole acrylic acid was added to a final concentration of 20 μg/mt, and A41
Each cell was shaken at 67°C for 2 hours. Control cultures were grown under the same conditions in the absence of β-enA. Culture qI/
) Centrifuge the liquid and remove the stinging liquid.
M loaded with 5% and ampicillin 25 μg/ml
MMJ was suspended in 9 medium. 50μL! 135s methionine was added to the whole culture and the cells were imaged for 30 min. After labeling, cells were pelleted and treated with 100 mM at pH 6.8 containing 5% glycerin Z and 1% sodium dodecyl sulfate (SDS).
) Lis(hydroxymethyl)aminomethane (Tris) 5
0μ was resuspended in it.

This AT cell suspension was boiled for 6 minutes to make Ze-1 into a pellet, loaded on a polyacrylamide gel for separation of 10% of total sample and addition of 3 samples, and electrophoresed at a constant current of -25 ma. According to Coomassie brilliant blue staining, there is a protein profile shift of approximately 29,000 daltons in cultures grown with β-FA compared to the protein profile of the same clones grown in the absence of β-FA. It is clear and necessary that one band is overproduced. The highest overproduction level occurred with clone pKGP44. The gel was dried and radiophotographed. β-I
In f@ cultures of pKGP44 that were not induced with AA, the labeling pattern was similar to pBR322.

It was evident that a disproportionate amount of labeling occurred in the 29,000 Dalton protein band in the induced J@ feed. β-lactamase enzyme assay (1) was performed before and after induction. The enzyme activity of β-lactamase was in direct correlation with the induction of the 29,000 Dalton protein. There is immediate evidence that tryptophan p,o, which is produced in E. coli, can direct protein overproduction in E. coli.

Subsequent experiments were conducted to determine the best possible conditions for overproducing β-lactamase using plasmid pK(J).
P 43, 44 and 45 were performed on E. coli. 0.5% b1 containing pKGP 45
of casamino acids and 25 μg/ml ampirin overnight in M9. i4d W xβ
-A1 of engineering AΔ. I was there for 411,7 days. Protein analysis of the induced culture showed a dominant band migrating at 29,000 daltons.

This band was not present in the pBR322 control.

Densitometer records showed that more than 80% of the new protein was β-lactamase (presumed precursor and mature protein). Coomassie blue staining of proteins from induced cells showed that more than 25% of total cellular protein was β-lactamase. DIIA sequence analysis of pKGP 45 predicted a P,O6 sequence.Published trp
It was shown to match the P, O1 sequence. ? J44
Figure A and Ce1l d” of Mr. 5iebenliet et al.
r Vol. 20, pp. 269-281 (1980) 'If I want to be illuminated.

Example 2 Plasmid purification, restriction enzyme digestion, sequential transformation and screening were performed according to the procedures described in Example 1.

Plasmid pLG 400 k P8°tl and Hl
Digested with ndlfl. Plasmid pKGP 36k
pst;I and HlndJII and purified the fragment containing the entire promoter.

The pKGP36 fragment was then ligated to the k pLσ400 digestion product and E. coli strain 112, LG99, was transformed using this ligated DNA'i. DNA from selected transformants f Birn, cited in AfJ.
It was prepared according to the method of Boim B and Doly and subjected to restriction digestion using PF3t J/H1ndl.
I got it. The digest was separated by electrophoresis on a 0.8% agarose gel and correctly fused plasmids were identified. One of these, pAP16, was selected for the wheat study. The analysis shows that 1 (pa I/H
The 1ndlll breakpoints are 39 bp apart and Hp
a ]/BamH] cut point (ri 45 bp apart, fc, see all of FIG. 5).

Example 3 Preparation of pAPPIF9 Plasmid 3847, a plasmid containing interferon cDNAi, was prepared by standard methods. cD
NA is Wicken Nishi et al. 9 J, J31o1. O
hem.

Volume 253, pp. 2483-2495 (1978) was synthesized using the general method of No. 471. cDN
The A arrangement is shown in FIG. Cloning is Vili
a-Komaroff et al., jl) Proc, N
atl.

Acad, Sci, USA Vol. 75 No. 3727-37
31 Sada (1978), especially Nos. 6727 and 6728
A standard method of writing was implemented. Plasmid 38
47 was digested with n1ncll and Bgtl, and the engineered Ftβ cDNA fragment was separated by electrophoresis on a polyacrylamide gel and then purified. H1nCIl/Bg
The tIf fragment (Figure 6A) was further digested with Hae III, which resulted in the mature coding sequence (Silk 6B).
1F-N-pcDNA total DNA at the 471st base of
Aru.

The 'H1nCfi,/Hagm1FN-β cDNA fragment was separated by electrophoresis on a polyacrylamide gel and then purified. H1ndllll linker (OAAGOT
TG)r H1ncll/Haeil engineering FN-βcDN
It was ligated to both ends of the A fragment.

The ligation product (Figure 6C) was digested with all Hindll and now each end Kgjndll I fltU I
The desired FN-β cDNA fragment containing all the components was separated by electrophoresis and then purified from polyacrylamide gel. Plasmid pAP 16 ([-)11n
Digested with dIII.

■P″N-β cDNA fragment ligated into vector and linked DNA-t?1)% m Ic-12
strain L (J-90k). Plasmid DNA was prepared from the selected transformants described above for the 61.5 product of pAP 16 and digested with 4 petl. This vector was transformed into one P6 strain. Since it has only one breakpoint and the IFN-β cDNA omits one Pstlw breakpoint, plasmids with two Pstl breakpoints (i-) were selected for further analysis.

Restriction mapping showed that the fragment was inserted in both possible directions. One recombinant plasmid, pA
PF9 contained the start codon of the FN-β CDNA fused in the correct orientation to the P,O,i fragment of pAP16. IFN-βCDJJA is fused within the framework of the macxz gene to constitute an IFN-ββ-galactosodase fusion gene. The opposite orientation was called pAPF1F8.

Further analysis showed that pAPFIF9, but not pAPF'lF8, produced β-galactosidase activity. In addition, pAPF'l:j, i'9 encoded a predicted fusion protein, but pAPF'l:j, i'9
I didn't give +1.

Example 4 Plasmids pAPFIF9 and pAP16 were engineered into FN
- i7. used to clone, edit and express β-ligation in E. coli. Ta. In order to express the active protein FN-β in E. coli, the DNA sequence encoding the 1:FN-β signal sequence was removed using the method of FN-β. Nucteic A by Mr. GoecLde et al.
c1dRee-'MA Volume 8 m4057-4074. R
(1980) modified Nyoshi, IF using the method described.
N-βc1) Boat-leg oligonucleotide ATGAGC on one strand of NAv-denatured H1ncn/BglH fragment
TAOAAC! f crossed.

K] of DNA polymerase], using the polymerizing activity of the t3n OW fragment and using oligonucleotides as primers to generate IFN-βPθt14 point k.
) A new chain was constructed with lu. Subsequently, in another development, 3'→5' exonuclease activity disrupted the signal region of the oligonucleotide and 1FN-βcL)NA, which terminated in the formed duplex. This modification of Goeddal et al.'s method is necessary to obtain sufficient yields of assembled fragments for cloning. After digestion of the reaction products and separation on polyacrylamide gels, the flanking 14[1 bp blunt-truncated Pst I band was purified and incorporated +F11 into the expression vector as follows.

Plasmid pAP16k was first digested with Hlndll and cut with Hind to give blunt ends. This reaction was digested with P8tl. Amino terminus of β-lactamase atom and U' I) KGP 36
P, O, -8, D, blunt edge Pst containing all fragments
One fragment was purified from polyacrylamide dal.

The PBt I fragment was digested with plasmid pAPFIF9 f Pst I and containing the carboxyl terminus of the β-lactamase gene and the carboxyl terminus of the FN-β0 DNA fused to the aac gene.

pAP16 Pθtl blunt truncated fragment, pAPFIF
9 Pstl fragment and edited Ps'tl-blunt cut ■ FN-β fragment The entire assembly of the king connection illustrated in Figure 7/c0 Escherichia coli was transformed using the molecular sound of 112aLG9Q all connected. . Properly assembled molecule ff, pAP
Due to the intoxication from the IFN-β/aac-Z fusion product obtained from F9, KM9 XgaA Play 1 appeared in color. If the structure of the atrocities is tlj (J translation cartography and D
Confirmed by NA sequence analysis. The diplar clones pK, GP 12-2 and pKGP13-19 were identified in this manner. Pst J from pAPFIF9
Fragment is IFN-β edited cDNA to pKGP56 prff motor? + Desired selection of Gukome that fuses exactly? Gave. A properly constructed molecule was described for all blue colonies.1) API6 plasmids.

Example 5 An alternative editing method to that described in Example 4 was used using pAPFIF9 and 9at31 echin nuclease. Cilasmid pAPFIF9 f was digested with Hpa I restriction enzyme. This resulted in five different W fragments, one of which contained the engineered FN-β portion 1r:@'1g of the fusion gene. These fragments were randomly digested with !7Bat3i exonuclease using standard methods. Collapse and BamHI
I digested it. For this operation, Jul) Engineering FN-β gene 5
'Randomly collapsed at the end and unique BamHf
A slightly delayed fragment with the wood end at the vJ break was obtained. Only the 5′-end deleted IFN-β T piece initiates translation/
It has an in-frame ATG and terminates at the EamF [cut point, which is the engineered FN-β fragment t:pAP of the frame Pe.
Let's fuse seven genes of 16 β-galactosidase. Vector pAP 16 was digested with Hindlll and the zigzag ends? The deoxynucleotide triphosphate was filled in with Klenow fragments of DNA polymerase to produce pure fragments. This blunt piece was then digested with M molecule f BamHI. Randomly collapsed engineering F
The N-β fragment was ligated into a modified pAP16 vector and the DNA was used to transform E. coli strains LG 90 and E. coli HBIOI as outlined in FIG.

The resulting clones (]-pKGP 12-2 and pK
GP13-191/ζ was screened as previously described. Clone pKGP 9-25 B deleted the pre-IFN-β sequence as predicted and pAP
A plasmid containing one ATG containing the mature ζ-active protein trace and another ATG fused to a single promoter fragment of 16 was present. All structures are DNA
Confirmed by sequence analysis.

Practical example 6 i9R and 9-25Rl/) preparation IFN-β extraction, K is aa c to obtain plasmid
Plasmid pKGP 131 fused to l Z gene
Edited IFN- from 9.12-2 and 9-25
The carboxyl terminal sequence of the β gene must be replaced with a functional carboxyl terminal sequence of the FN-β gene. All three labeled fusion plasmids were engineered in the same manner.
It was transformed into a β expression plasmid. Only the transformation of pKGP 13-19 will be described below.

The PstJ/EamH engineering fragment from pBR322 was purified.

This fragment contained the carboxyl terminus of β-lactamase and the entire origin of replication of pBR322.

P of pKGP '36 fused to the 7-mino terminus of β-lactamase and the edited amine terminus of engineered FN-β,
0. (PstlK from pKGP13-19 containing H
The fr piece was purified. Finally, Pstl/Egt...carpogysyl-terminated IFN-β fragments were assembled by three-way ligation of all these six fragments (see Figure 9), and the entire DNA was used for transformation of MM294. . Plasmid DNA f? Digestion with Pet 1 with Ja perilla followed by analysis on an agarose gel. A plasmid with two Pθtl breakpoints separated by approximately 1000 base pairs? ! -@ltq clones were sequenced and assayed for TFN-β activity. The DNA sequence was selected and carried out by cloning the fragment into M15 phage DNA and the DNA sequence fC New England Biolabs.

It was determined by the dideoxy method using sample mulch that could be supplied by.

To assay for FN-β activity, %X111 bacteria were grown to an optical density of 1 and 20 μg/m
By oscillating f'JJ at about 2 o'clock in the presence of β-■AA of l,! lll induced. (Concentrated +-100 times and lysed by sonication. The cell waste was pelleted and the supernatant liquid k. J of Knight et al.
, Interferon Res, Volume 2, No. 421-
Inhibition of monocysotic degeneration effect described on page 429 (1982) 101
'' assay for antiviral activity. Plasmid pKGP12-2Rs 13-19R and 9-2
5Rk:L had a predicted truncated DNA sequence (see Figure 10) and exhibited antiviral activity. Antiviral activity is resistant to pH 2, sensitive to heating at 56°C, and F'N-β! Neutralized by specific antiserum. Therefore, this example demonstrates how to engineer FN in E. coli using the described cloning vector and expression vector.
- Demonstrates the cloning and expression of β.

Example 7 The transcription of the EFN-β gene on plasmids pKGP 12-2Rs 9-25R and 16-i9R is directed by a capable hybrid promoter of plasmid pKGP56. In order to perform efficient transcription, the tritophane promoter was regenerated in a two-culm cloning experiment.Plasmid pK
GP 43 is fully 1trp as described in Example 1
Contains P,,O,. Restriction endonuclease Hp
a, 1 cleaves the entire 110 region of trp b and pKGP 36 promoter. Plasmid pKGP 43
was first digested using the restriction endonuclease H1ndlll. The zigzag end of the Hlndlll was filled with two blunt end pieces by V straining as described in Example 4. Next, this L [piece was digested with Hpal, and the 165 region of the trp promoter was digested with Hpal. Contains H
The pal/H1ndllll filling fragment was isolated. Plasmodium pKGP 13-19Rt I (whitened with pa I) containing 135 regions of the trp promoter.
The aJ/H1ndlll filling fragment from GP 45 was ligated to the Hpal breakpoint of pKGP 13-19R and this DNA i was used to transform E. coli HJ3101.

Mini-lysates were prepared from selected clones and the orientation of the inserted fragment was determined. DNA'i from clones with the correct orientation was sequenced. 1 in sequence analysis
It was confirmed that the ridge clone pKGP 15-19R-trp6 contains a rearranged trp promoter fused to the desired engineered FN-β gene (see Figure 10). pKGP 16-1 pKGP 16-1 (11 cell degeneration effect inhibition assay was performed using the method described in the above-mentioned article by Knight et al.
9R and pi', OP 15-19R-trp6. The positive pKGP 13-19R culture contained approximately 5 x 1041 t/l of antiviral activity. A culture of pKGP 13-19R/shirp6 had an antiviral activity of approximately 107 μ/l. This clearly demonstrates the utility of the pKGP 36 promoter for gene cloning and expression experiments. This system should prove invaluable for the cloning and manipulation of foreign gene sequences that, when expressed, cause instability of the recombinant plasmid in culture.

Example 8 ptH4, a plasmid containing engineering L-2 CDNA
The interferon cDNA of Example 6 was prepared by standard methods as described for the plasmid, plasmid 5847.

In order to express the active mature engineered L-2 in E. coli, the engineered L-2 cDNA id was edited to remove the 5' DNA coding region for the signal sequence and edited 1, L-2a DNA must be inserted into an expression vector. Plasmid ptH4
0 total Pstl and purified the cDNA insert. A Hg1A1 restriction site remains at the junction between the last amino acid of the signal sequence (Ser) and the first amino acid of mature engineered L-2 (Ata). Engineering L-2
The Pstl fragment containing the entire cDNA was cleaved with Hg1A1. A 1enOW fragment was used in DNA polymerizer 70 to remove 4 bases and obtain blunt ends, resulting in a 6' overhang of Hg1A11"i 4 bp. This operation removed the Ale, codon and matured A blunt end was added starting with ca'r, the codon for the second amino acid (Pro) of L-2.

To provide a codon for the start codon (ATG), a special adapter molecule (AJ) was devised and synthesized.
AATAOcGA has several valuable features. This is not only the AUG start codon, but also the first codon f of mature engineering L-2! :give. CG overhangs reconstructing the C1thl and Hina111 breakpoints were inserted into the vector as described below. This adapter molecule was ligated to the blunt end/Pstl cDNA fragment and the ligated 1) NA
Digested with Stu III and Stu 1. H1ndllll
/5tul 450 bp lfI fragment was purified on a whole polyacrylamide gel and the purified fragment was purified on a p[3R3
22 2625 bp H1ndll/PVJ fragments. This I-gradient was possible because restriction with 5tul and Pvul gave blunt ends. Then DN
A ligated canine intestine was used for conversion into the form of M294. plus ε
FDNA was selected and purified from C clone.
ba] (one XbaI breakpoint remains within the engineered L-2 cDNA sequence). One vector, pBRBIL-2, has Xba 11 in the predicted position.
) J-point 4 words E a-L were selected to further compose them. The stability of Baekku-pBRE engineer L-2 was increased because the 几-2 gene lacks an active promoter. This backside is shown schematically in FIG. 14.

Spindle pKG of vector pKGP 56 (Example 1)
In order to promote the fusion of the P36.trp u IL-2 gene to the trp promoter, it was prepared as follows. pKGP 36k was digested with Hpal/Pstl and purified as a large fragment (3640 bp) containing the entire carboxyl terminus of the tθtR gene, origin of replication, and β-lactamase gene. Vector pKGP 1
3-19R-trp 6 (Example 7) k Hpa
/JS small fragment (1531] bp digested with l/Pstl and containing the amine terminus of the β-lactamase gene.
) was purified. All of these 2 m fragments were ligated and the DNA was used to transform E. II bacterium MM294.

A clone containing the entire plasmid gene of one Hpa I 9J 19r point was selected and used for further studies.

Plasmid DNA was purified from one clone and t
Complete trp promoter fusion was confirmed by rearranging the DNA'i sequence in the rp promoter region. This vector was called pKGP 56 trp.
p (approximately 6.0 x 106 Daltons).

The entire trp promoter was added to the plasmid pK as follows.
It was fused to the engineered L-2 gene using GP36.trp and pBREJL-2. Vector pBRI
nIL-2i was digested with H1rollL/Pstl and contained a large (2000b'
p) Purification of IliQ pieces. Plasmid pKGP36・
trp f ) (indlll/, digested with Pstl and a small fragment containing the trp promoter (153
0bp)'t-purified. The two citrus fragments were ligated and the DNA was used to transform E. coli MM294. Clocks containing plasmids of the predicted size with one Hpal and Xba I ffTII restriction site were selected for further analysis. 1 clone D
NA sequence analysis confirmed that a fusion of the trp promoter and the engineered L-2 gene had occurred. pTrpEixL=2, see Figure 15. II, -2
Total protein from E. coli with and without the expression vector was separated by polyacrylamide gel electrophoresis. At 15, a novel protein that migrated with Dalton's apparent molecule 'M was present in #I cells containing the expression vector but not in control cells. This protein migrated to the VC as expected for non-glycosylated L-2. Lysates of cells containing pTrp[IL-2 were positive and control cell lysates were negative in a radioimmunoassay using antibodies against native IL-2.

T-L-2 activity is a cloned murine cytotoxic T
-Lin/Zero lineage (OTLL-2, subclone 15H
), L-2 concentration-dependent proliferation stimulation (measured with 5H-thymidine) was determined in D3+11.

Extracts of cells containing pTrpK-L-2 were cultured in culture 1.
The control extract containing pKGP36.trpf did not produce any activity, while the control extract containing pKGP36.trpf produced 0 to 5000 relative related units. Antibodies against native IL-2 neutralized the activity in the recombinant extract. This demonstrates that the two genes inserted into the expression vector directed high levels of silk protein production in human L-2.

[Brief explanation of the drawing]

Figure 1 shows the vector pAOYc! 177, a conventional method plasmid pLD 102'l containing a tryptophan-deleted trp D102 sequence that fuses the O gene of the ivy and tryptophan operon to the tryptophan leader ribbon genome integration site: shown. FIG. 2 shows the conventional positive pBR322 system-r0. FIG. 3 shows P, 0. Figure 4 shows the restriction endonuclease map of the novel plasmid pKGP 43 containing the entire β-lactamase gene with the orientation being that of the β-2 lactamase gene. The iJA diagram is P, 0. is tetracycline resistance (tθt
R) Restriction endonuclease map of the novel plasmid pKGP66 containing the oriented β-lactamase gene. 4th Bme 1 is a new P from pKGP 36, 0. Comparison of fragments versus trp P, OJf+ fragment fc shown T. Figure 5 shows pKG fused to the β-galactosidase gene.
P from P 36, 0. A new plasmid p that n@
Figure 6 shows the restriction endonuclease map of the parent plasmid pKGP 12-2 j?
and the restriction endonuclease map of pKGP 15-19, and the pAP16 Pst I blunt end fragment,
pAPF engineering F9Pst l fragment and edited Pet
1 shows their preparation using three-way ligation of blunt-ended 1FN-β fragments. FIG. 8 shows an alternative editing method for the preparation of plasmid pKGP 9-25 using pAPFIF9 and BaA31 exonuclease and ligating into pAP 16. Figure 9 u 1) Pstl/BamHI from BR322
Fragment - Pet 1 fragment from pKGP13-19 and P day tl/Egt... carboxyl-terminal iFl+-β fragment between kings? : New plasmid to be ligated I) KGPl
Preparation of 6-19H. Figure 10 shows the plasmid pKuP 13-19R.
The digestion, ligation and transformation steps to form trp6 are shown. FIG. 11 shows the cDNA sequence of engineered FN-β geneticist from plasmid p5847. FIG. 12 shows the plasmid pKGP 12-2 of the present invention. R
-pKGP 13-19R and pKGP 9-25R
The DNA sequence of the transcription unit containing the P, O, -S, D, fragments characterizing 1' is shown. Figure 13 shows a 400 base pair fragment containing the Hpa1 breakpoint from pLD102. Figure 14 shows the larger k obtained by restriction of the entire edited L-2 gene fragment pBR322 from pIH40.
Figure 2 shows the preparation of pBREIL-2 vector by ligation to the l in dIIL'Pv uII fragment. Figure 15 shows pKGP36 trp and pTrpH:
The latter pTrpgIL- by ligation to a fragment from pBREIL-2 containing a portion of the L-2 gene and a portion of the ampicillin gene. 2 is shown. FIG, 1 PO delusion vector unidirectional FIG, 6 IFN-βcDNA/HincTr ATGATGBg
lII AHindrII Liquor 25 strength / 1lind
+41 ”G ATG 1(indHI C+ FI6.9 NIP2NH2 pKGP13-19 PstT PstIAMP I
FN -8 + 0OH p3847 Pstl Bgll TFN-1+ + Riyu, 1φ purchase conversion p1840 F / G, /4 ↓ PstI included 5nserAlaProThr 5tuf
f Pstff3'+5'EXO↓ MetAla ProThr 5tuff Pstl oriconucleotide Attaph9terFIG, 15 Continuation of page 1 ■InJC1,4 identification symbol Internal serial number priority claim
01984 July 5 [phase] United States (US) 06280 departure
Written by: Stephen Ot<-42 Meninding Building Drive, Hockessin, 19707

Claims (1)

Claims: 1) Consists of approximately 600 base pairs and contains a 110 region and a trpi lomotor oscillator Shine-Da.
Shine-Da1ga showing characteristics of 1garno fragment
Promoter-8hi with rno sequence
ne -DalgarnO01ai-containing fragment, (1) TGC in the -65 region of the fragment (5'→6')
! OGA 0GGOT, and (II) the first 8 base pairs (5'→3') are 0.
A fragment having an improvement consisting of a sequence of about 17 base pairs between the -65 and -10 regions of said fragment which is GCGOATO GOGOGTAG. 2) A plasmid containing the fragment according to claim 1. 3) 1. (i) the gene encoding β-lactamase; or (II) the gene encoding engineered FN-β (β-interferon) and β-lactamase. The plasmid described in Section 2. 4) Contains the gene encoding β-lactamase and the gene encoding tetracycline resistance, the promoter-o and the promoter-o are oriented in the direction of the tetR gene, and the molecular weight of the cilasmid is approximately 3.0x10
6 daltons, and the plasmid has the following partial restriction map: Pstl-1coRI, 750'bp;
KcoRl-01al, 23bp; C! mal-HP
al, 565bp; C1al-clal, 6oobp
:Hpal-01al, 35bp; Hpal-Hln
dl [, 39 bp; Hlndll-BamHI, 34
5 bp (wherein bp refers to a base pair) of the plasmid pHP36.5) according to claim 3, which contains the β-galactosidase gene lacking the initiation ATG codon, and the promoter-O is the promoter of the gene. The molecular weight of the plasmid is approximately 4.8X1.
0' Dalton and the plasmid has the following restriction map: PstJ-Hlnd[l, 1300 bp;
01al-01al, 600bp: Hpal-Hln
dll[, 39bp: Hpal-BamHl, 45b
6) Plasmid pAP16 according to claim 2 having a fusion gene encoding β-galactosidase and the amino terminus of engineered FN-β;
The gene encoding β has been edited so that its signal sequence has been removed, and the promoter-O is oriented in the direction of the edited engineered FN-β gene, and the molecular weight of the plasmid is approximately 5. lX1
0' Dalton and the plasmid has the following restriction map: Petl-Hpal, 1320 bp; Hp
al-01al, 27bp; 01ai-Pstl, 1
45bp; Petl-Hlndll, 270bp;
Hlndll-BamHl, pKG with 6 bp
P12-2. pKGP13-19 and pKGP9-2
5. A plasmid according to claim 2 selected from the group consisting of 5. 7) Contains the gene encoding the biologically active protein IFN-β, with the promoter
The direction of the β gene is oriented, and the molecular weight of the plasmid is approximately 3.
．． (
; HpILI-0'lal, 27bp: 01al-
Pstl, pKGP12-2 with 145tlp
3. A plasmid according to claim 2 selected from the group consisting of R, pKGP13-19R and pKGP9-25R. 8) A trizotophane promoter containing the gene encoding β-lactamase and the gene encoding tetracycline resistance, and further oriented towards the β-lactamase gene.
n e -Ihlgarn. sequence, and the molecular weight of the plasmid is approximately 2.8×
106 daltons, and the plasmid has the partial restriction map described above, ie Pstl-Kco River. 750bp; FicoRl-Hpal, 101]bp
; I(pal-Hlndlll, 300bp: Hl
ndlll-BamHI,Ro45J)'f plasmid pKGP4 containing the gene encoding biologically active IFN-β, the plasmid containing the tricytophane promoter-oriented towards the EFN-β gene. Operator Shin e-Da 1 gar n'o sequence, the molecular weight of the plasmid is approximately 3.7 x 10" Daltons, and the plasmid has the following restriction map: Petl-Hpal, 153Qbp; Hpal-
01al, 27bp; C1al-Pstl, 145b
Plasmid with pKGP1319Rtrp6
. 10) A plasmid transformed with the plasmid according to claim 2. 11) Escherichia coli (B, coll) bacteria 12X transformed with the plasmid according to claim 2
l) restriction of the pLD type 102 plasmid with Hpal and Ba1N; (ii) isolation and purification of the Hpa[/Ba1l fragment containing the trp-promoter-o-retar;
1i) Modify the Hpa[[/Ba1l fragment by limited digestion with Taq l and (ψ) convert the above modified fragment into C1al of pBR322
A method for preparing a plasmid selected from the group consisting of pKGP43 and pKGP36, comprising the step of inserting into the breakpoint. 13) (i) 1) Restrict the BR322 type plasmid with Pstl and BamHl to Pstl/BamHI
A fragment was obtained and the fragment was purified, (li) (a) pKGP type 12-2, (b) p
KGP type 13-19 or (C)1) KGP9-25
The type plasmid was restricted using StK and β-2
Amino terminus of tamase, pKGP36 P, 00-8
．． D, (promoter-operator Shino-
The fragment containing the amino terminus of the edited nrN-71 gene was purified and the plasmid of type (iiDp3847 was restricted with Pstl and Bgl[ and the edited i+'N-β gene and 4v) step (1), (1υ and m(li) =>
, (b) or (C) plasmid pKGP 12-2R%pKG
Preparation of Pi5-19R and pKGP 9-25R. 14) (1) pKap type 43 plasmid into Hpa
l and Hlndl [and Hlnd
ll, filled with cutting point) Iinall
A fragment having the Hpal end and the Hpal end was obtained, and the (It) pKGP13-19R type plasmid was transformed into the Hpal end.
al t: to obtain the Hpa) fragment and 0
11) Plasmid pKGP 13-19R-tr consisting of the step of ligating the fragment of step (1) and the fragment of step (11)
Method for preparing p6. 15Xl) I) LG 400 type plasmid as Pst
Pstl/f(
(II) restriction of the cilasmid of pKGP36 type with Pstl and Hlndll and isolation and purification of the fragment containing the promoter-operator and G11) promoter-operator of pKGP36.
A method for preparing plasmid pAP16, comprising the steps of ligating the containing fragment to the Pstl/H1ndlll fragment of pLG400. 16 Xi) growing the bacterial cells to a specified optical density; (11) fixing the cell wall by treatment with a calcium chloride solution; and 011) mixing the competent cells with the ligated DNA mixture containing the plasmid. A method for modifying a bacterial microorganism to include a plasmid as claimed in claim 2, comprising the steps of: and culturing the mixture to effect cilasmid insertion. 17) A biologically pure culture of a microorganism according to claim 10. 18) A biologically pure culture of a mutant of the microorganism according to claim 10, which is capable of expressing β-2 tamase or β-lactamase and engineered FN-β. 19) The plasmid contains a gene encoding β-lactamase and a gene encoding L-2 (interleukin-2), and the molecular weight of the plasmid is approximately 1.7.
X10'dalton and whose plasmid has the following partial restriction map: Hl containing the engineered L-2 gene.
ndlll-IL-2 day tul breakpoint, 450 bp
1 L-'l5tul breakpoint-H1n (Cilasmid pBRK L-2゜20 with LIII 2325bps) that does not contain the L-2 gene and encodes the gene encoding β-lactamase and tetracycline resistance The trp promoter is oriented in the direction of the tθtR gene, and the molecular weight of the plasmid is approximately 3.0.
X10'dalton and its plasmid has the following partial restriction map: Hlndl[1-Petl 358
61) plPstl-C1a1775bp, Trp
01al-01a1790bp including P, O,
, 01al-Hlndlll 4bp t-plasmid pKGP36.trp. 21) The plasmid contains a gene encoding β-lactamase and a gene encoding L-2, and the molecular weight of the plasmid is approximately 2. lX10” Dalton,
The cilasmids are shown in the partial restriction map below, i.e.
IL, containing the -2 gene t'leH1ndllll-Pe
Pstl containing tl 2000bp, Trp P,o, (tryptophan promoter-operator)
- Plasmid p with HlnaIll 1569 bp
TrpKIL-2゜22) A microorganism transformed with the cilasmid according to claim 2.1. 23XI) pBRg engineering L-2 type plasmid into Hln
Limit and set using all and Pstl f.
A fragment containing the 2R gene was prepared, and a (It) pKGP36-trp type plasmid was ui
limit using nalfl and pstl f and tr
p P, O. 22. A method for preparing a plasmid according to claim 21, comprising the steps of: (iii) ligating the fragments of step (1) and step (ii). 24) A biologically pure culture of a microorganism according to claim 22. 25) A molecule (A) that includes the initiation codon that is the first diton of mature L-2 and has the sequence 0GATAAGOTTATGGOT TATTOGAATACOGA.