Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/495—Transforming growth factor [TGF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

• the invention relates generally to recombinant plasmids for expressing proteins that are secreted into periplasm or culture medium. More particularly, the invention relates to the construction of plasmids which, when introduced into an expression vector secrete proteins directly into the periplasm or extracellular culture medium in which the vectors are grown.
• Proteins that are secreted into the periplasm of E. coli or inserted into E. coli membranes have several unique features. Nearly all proteins destined to be exported have a signal sequence (SS) which has been shown to be important for translocation. In addition, membrane or secreted proteins have structural features within the molecules that facilitate secretion or membrane insertion.
• SS signal sequence
• membrane or secreted proteins have structural features within the molecules that facilitate secretion or membrane insertion.
• the construction of chimeric proteins have been studied in which portions of the Pseudomonas exotoxin (PE) gene are fused to other functional peptides and which are directed by the toxin to receptors on target cells. The chimeric genes are expressed in E. coli and the chimeric proteins are purified from the bacterial cells.
• PE Pseudomonas exotoxin
• X-ray crystallography of mature Pseudomonas exotoxin has shown that the molecule is composed of three distinct structural domains. Various portions of the Pseudomonas exotoxin gene have been deleted and expressed in E. coli in order to analyze the function of these domains. Study of the expressed mutant proteins has shown that the amino terminal domain I having amino acids 1 through 252, is responsible for receptor binding, the middle domain II having amino acids 253 through 384, is responsible for translocation of the toxin across membranes, and the carboxyl terminal domain III, having amino acids 385 through 384, contains the ADP-ribosylating activity.
• Pseudomonas exotoxin is secreted by Pseudomonas aeruginosa into its growth medium.
• the gene of this article is not related to a gene from a Pseudomonas exotoxin.
• the industry is lacking an expression vector or plasmid having a Pseudomonas exotoxin that, when introduced into an appropriate host, causes a desired protein to secrete into the periplasm and/or the culture medium of the host cell in which the vector is grown.
• the industry is also lacking a biological method for synthesizing pure or fusion proteins in large or commercially acceptable amounts which uses an expression vector or plasmid having Pseudomonas exotoxin.
• the invention includes an expression vector that expresses a protein that secretes from a host cell into the periplasmic space and/or a medium of the host cell.
• the expression vector comprises a gene portion or polypeptide segment for expressing at least amino acids 381 through 407 of Pseudomonas exotoxin and a gene for expressing the protein that is to be secreted from the host cell.
• the gene is ligated near or to the first gene or to a gene providing a cleavage site between the protein and the Pseudomonas exotoxin amino acids.
• the expression vector can include a signal sequence.
• the invention includes the method for synthesizing pure protein from a host cell having an expression vector containing a gene for the desired protein and a gene portion for expressing at least a portion of domain II of Pseudomonas exotoxin.
• the invention includes the expressed and secreted fusion or pure protein products of the invented process.
• Figure 1 represents various Pseudomonas exotoxin derivatives and fusion proteins.
• Figure 2 represents cellular localization of Pseudomonas exotoxin derivatives by immunoblotting.
• a plasmid comprising a promoter, optionally, any outer membrane signal sequence attached to a gene for a protein which is desired to be secreted, and a PE40 gene required for translocating across membrane.
• polypeptide segment in reference to domain II of Pseudomonas exotoxin refers to any consecutive group of amino acids from this exotoxin that increases the secretion of a protein to which it is fused.
• Such polypeptide segments can include all of domain II of the Pseudomanas exotoxin.
• polypeptide segments can include a portion of domain I and/or III of this exotoxin along with a functional portion of domain II.
• gene portion refers to a segment or nucleotide sequence that encodes a polypeptide segment of domain II of the Pseudomonas exotoxin that increases the secretion of a protein to which the polypeptide segment is fused.
• the desired protein to be secreted is referred to as the "selected protein.” Molecules containing domain II of Pseudomonas exotoxin are secreted into the periplasm of E. coli and under certain conditions are also secreted into the medium.
• PE40 A molecule termed "PE40" which contains both domains II and III of Pseudomonas exotoxin is secreted into the periplasm without an OmpA signal sequence present and into the medium when the signal sequence is added. This result suggests that sequences within domain II in concert with a signal sequence can promote secretion of a protein into the medium. However, the deletion of all of domain I of Pseudomonas exotoxin and a portion of domain II also generates molecules that are secreted into the medium and periplasm without a signal sequence. In addition, the placement of alkaline phosphatase at the carboxyl terminus of a toxin molecule containing domain II alone or domains II and III also leads to secretion into the medium.
• Domain II contains a large number of hydrophobic amino acids.
• the amino terminus of each of the recombinant Pseudomonas exotoxin proteins can be constructed to contain the sequence met-ala-glu-glu. This amino terminus in the invented constructs is different from a conventional leader sequence which must possess positively charged amino acids in the amino terminal region. It has been reported that introduction of some random sequences in place of a signal sequence could lead to the secretion of invertase in yeast, but the results of the invention suggest that sequence within domain II of Pseudomonas exotoxin are important for secretion of molecules across E. coli membranes.
• domain II When domain II is combined with a signal sequence such as that found in OmpA or with a protein which itself can be transported across a membrane such as alkaline phosphatase, secretion into the periplasm and into the medium results. Furthermore, a protein such a TGF-alpha can be efficiently transported into the medium or periplasm when fused to domains II and III of Pseudomonas exotoxin.
• Domain II is composed of a series of alpha helices. Stretches of hydrophobic amino acids are present in these helices. Domain II is also required for efficient translocation of Pseudomonas exotoxin into the cytoplasm of animal cells during endocytosis.
• Domain II of Pseudomonas exotoxin has an essential role in the secretion of the toxin into the periplasm and medium. Domain II can also promote the secretion of other molecules fused to it. Domain II has been examined by expressing modified Pseudomonas exotoxin genes in E. coli under to control of a T7 promoter. Without a signal sequence, Pseudomonas exotoxin accumulates within the cell, but it is secreted into the periplasm when part or all of domain I is removed.
• Pseudomonas exotoxin appears in the periplasm and medium when domains I and part of II are removed. Domain II alone is secreted into the periplasm, whereas domain III alone remains within the cell. The addition of an ompA signal sequence results in the secretion of mature Pseudomonas exotoxin into the periplasm and secretion of domain II-III into the medium.
• a protein composed of a transforming growth factor alpha, fused to the amino end of domain II-III, is secreted into the periplasm without a signal sequence and into the medium with a signal sequence.
• a protein composed of domain II or II-III fused to the amino end of alkaline phosphatase is secreted into the periplasm and the medium with or without a signal sequence.
• the preferred embodiment of the invention uses Pseudomonas exotoxin in an E. coli system, although expression vectors other than E. coli can also be used. A portion of the Pseudomonas exotoxin gene is fused to the gene of interest.
• the protein is expressed in E. coli using a T7 late promoter expression system and an E. coli outer membrane protein signal sequence such as that of ompA, the chimeric protein is secreted into the medium in large amounts. If ompA signal sequence is not present, the chimeric protein is secreted into the periplasmic space.
• Table 1 The results produced by ligations of various combinations of these elements are shown in Table 1.
• T7 promoter/ompA/protein x /PE40 Medium T7 promoter/ompA/PE40/protein x Medium T7 promoter/PE40/protein x Periplasm T7 promoter/ompA/protein x Cytoplasm or Periplasma T7 promoter/protein x Cytoplasm
• PE Pseudomonas exotoxin
• the PE40 protein secreted into the medium has full biological activity and has a native structure. Usually foreign proteins made in E. coli accumulate in an aggregated form and must be denatured and renatured to yield an active protein, and then must be purified free of other E. coli proteins.
• PE40 is secreted into the medium in an enriched and native form. Transport of fusion protein into periplasm can occur as follows. A cDNA for the transforming growth factor alpha (TGF-alpha) has been fused to the gene encoding PE40. The TGF-alpha gene has been placed at the 5' or the 3' end of the gene encoding PE40.
• TGF-alpha transforming growth factor alpha
• TGF-alpha-PE40 When placed at the 5' end (pXY 382) a fusion protein, TGF-alpha-PE40, is produced and some of it is transported into the periplasm.
• a cDNA for the interleukin 2 gene can also be placed at the 5' end of PE40 (pHL 310) and it too is transported into the periplasm.
• TGF-alpha fused at the 3' end of PE40 (coded by pVC33) is also transported into the periplasm, but the protein is degraded. Degradation of this protein may in part be due to the amino acids used to link PE40 to TGF-alpha.
• Excretion of a fusion protein into the medium occurs as follows.
• the gene encoding the TGF-alpha-PE40 fusion protein also contains an ompA signal sequence at its 5' end, the resulting protein is secreted into the medium in large amounts . More than 30% of the protein produced is found in the medium in an active form with 20% transported to periplasm.
• Fusion genes in which genes encoding other proteins are fused to the coding sequence for Domain II alone or to Domains II and III or Pseudomonas exotoxin with a signal sequence are similarly constructed. Such genes are, for example, interleukin-2 and alkaline phosphatase.
• Any gene encoding a protein that is usually transported across membranes of cells can also be secreted into the medium in accordance with the methodology of the present invention.
• Numerous other genes for expressing and secreting desired proteins can be selected for use with the invention.
• Such other selected proteins can include Staphylococcal nuclease, gal repressor, bacterial Cytoplasmic, Cyclic AMP receptor protein, bacterial Adenylate Cyclase and human soluble T40 receptor (CD4).
• Pure proteins can be made from fused proteins produced by fusion genes by providing a site into the fusion protein that can be cleaved by a specific protease or a specific chemical reaction.
• An example of such a site is cleavage by the enzyme collagenase. Collagenase splits at gly-leu or ile-ala-gly bonds which are found in collagen, but are rare in other proteins. Elastase is another such agent.
• the candidate fusion protein does not contain an internal methionine residue, a codon for methionine can be created in between the candidate protein and the Pseudomonas exotoxin domains and cyanogen bromide can be used to generate the desired protein. Similar other strategies well known to one of ordinary skill in the art can also be used. Deposits have been made at the ATCC, Rockville, Maryland, on September 19, 1986, under the following accession numbers:
• the deposits shall be viably maintained or replaced if they become non-viable, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law.
• the Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
• pVC4 was cut with Hindlll and Tth111l, treated with SI nuclease and the large fragment ligated to itself.
• pVC20 pVC4 was cut with Hind III, treated with Klenow fragments of DNA polymerase I, and cut with Pstl to yield a 1.6 kb fragment.
• pVC4 was cut with Bglll, treated with SI nuclease and then with Pstl. A 2.1 kb fragment was isolated and ligated to the 1.6 kb fragment to produce pVC20.
• pVC85 pVC8 was treated sequentially with Ndel, S1 nuclease and Xbal and 3.6 kb fragment isolated.
• pINIII OmpA1 was treated sequentially with EcoR1, Klenow fragment and Xbal and 92 bp fragment, carrying an ompA Shine-Dalgarno ( SD ) region and an ompA signal sequence was isolated.
• the 3.6 kb fragment and the 92 bp fragment were ligated to produce pVC85.
• Other plasmids with OmpA signal sequences were created similarly.
• the phoA containing plasmids were constructed using a Pstl insert that contains the phoA gene.
• pVC229 was constructed by treating pVC8 with Sst II followed by T4 DNA polymerase to yield a 3.65 kb fragment that was dephosphorylated.
• pCH39 was treated with Pstl and T4 polymerase.
• a 3 kb fragment was isolated, ligated to the 3.65 kb fragment and the recombinants were screened for proper orientation.
• pVC2295 was constructed from pVC85 similarly.
• pVC809 was constructed by treating pVC8 with PpuMI followed by Klenow fragment to yield a 3.65 kb fragment.
• pCH2 was treated with Pstl and T4 polymerase to produce a 3.0 kb fragment. The two fragments were joined and recombinants checked for proper PhoA orientation.
• pVC8095 was made from pVC85 similarly. Construction of pXY382 and pXY3825 is described below. These contain a TGF-alpha cDNA fused to the 5' end of the DNA for domains II and III of PE. pXY3825 also carries on ompA signal sequence. In all the plasmids, the genes are linked to a phage T7 late promoter. Other plasmids and bacterial strains were previously described. Plasmid construction is further understood with reference to Figure 1. Figure 1 is a representation of various Pseudomonas exotoxin derivatives and fusion proteins.
• Plasmid pXY382 contains a TGF-alpha cDNA fused to the 5' end of domains II and III of Pseudomonas exotoxin.
• the circled number "50" is the last amino acid of TGF-alpha.
• pVC229 and 809 contain the phoA gene fused to the 3' end of the gene for domains II or II and III.
• the plain numbers indicate Pseudomonas exotoxin amino acids.
• the boxed number (6, 14) indicate PhoA amino acids. Gly and ala were added during cloning.
• pVC2295 and pVC8095 are similar to pVC229 and pVC809 except for an ompA signal sequence.
• E. coli BL21 (lambda DE3) cells carrying a desiredplasmid were grown and induced with IPTG and described above. After induction, the culture of 30 ml was centrifuged at 3000 rpm for 10 minutes. To obtain periplasmic and spheroplast fractions, the cell pellet was suspended in 1.5 ml sucrose solution of 20% sucrose w/w, 30 mM Tris, pH 7.4, 1 mM EDTA, kept on ice for 10 minutes and aliquots were removed for assay. The remainder was centrifuged at 6000 rpm and the supernatant of sucrose wash discarded.
• Examples 1 through 8 and Comparative Examples A and B illustrate the localization of Pseudomonas exotoxin molecules with various portions delated.
• Comparative Example A pVC4 encodes the entire Pseudomonas exotoxin protein with a methionine placed before the alanine at position 1. It was previously found that when the Pseudomonas exotoxin gene is expressed in E. coli, the toxin accumulates within the cell. Table I shows that over 98% of this molecule is found associated with spheroplasts. Proteins with deletions of progressive amounts of domain I encoded by pVC15, 19 and 20 were mostly found associated with spheroplasts but significant amounts appeared in the periplasm.
• Example 4 using pVC8 98% of the Pseudomonas exotoxin synthesized was secreted into the periplasm. This is further illustrated in Figure 2A.
• the protein produced by pVC8 has a Mr of 40,000 and hence is referred to as PE40. Deletion of increasing portions of domain II as in pVC9, 10 and 17, caused about 9% of the protein to appear in the medium with rest mostly appearing in the periplasm.
• Comparative Example B using pVC7 the resulting protein which consists only of domain III remained within the cell.
• pVC17 only contains 27 amino acids that are not present in pVC7 of Comparative Example B. These amino acids are very important for secretion.
• Examples 9 through 13 illustrate the effect of the ompA signal sequence on secretion.
• Signal sequences present in proteins of gram negative bacteria have been found to promote secretion of proteins into the periplasmic space or insertion of proteins into cell membranes.
• the effect of an ompA signal sequence on the cellular distribution of various molecules derived from Pseudomonas exotoxin is, therefore, presented.
• Table III shows that the ompA signal sequence present in Example 9 using pVC45 promotes the secretion of full length Pseudomonas exotoxin into the periplasm, but not into the medium.
• a similar result was recently reported by Douglas et al. (1987), J. Bacteriol 169:4962-4966.
• the ompA signal sequence apparently overcomes the inhibitory action of domain 1.
• the OmpA signal sequence had very little effect on the secretion of molecules in which various portions of domain I were deleted and are already secreted into the periplasm. For example, pVC155 and pVC205 with deletion of amino acids 4-137 or 5-224, respectively.
• the OmpA signal sequence did cause secretion into the medium of a molecule containing only domains II and III. This is further illustrated in Figure 2B. Addition of an ompA signal sequence also resulted in the secretion into the medium of domain II by itself as shown in Figure 2E.
• an ompA signal sequence precedes a protein composed of domain II alone or of domain II and III, the protein is secreted into the medium.
• the Pseudomonas exotoxin molecules present in the periplasm and in the medium are slightly smaller than the molecules in the spheroplasts indicating removal of the signal sequence as shown in Figure 2B and E.
• Purified PE40 from the medium of cells producing it has the N-terminal sequence of ala-asn-leu-ala. This indicates that the correct processing of the signal sequence.
• These examples demonstrate that the protein produced by pVC105 which lacks all of domain I and a portion of domain II was also secreted into the medium when the ompA signal sequence was present.
• Beta-galactosidase was measured in all experiments and ⁇ 1% of total activity was found in the medium indicating lysis had not occurred.
• the results of Examples 9 through 13 are presented in Table 3.
• Examples 14 and 15 illustrate the secretion of chimeric protein TGF-alpha-PE40.
• a chimeric protein is made by fusing a cDNA encoding TGF-alpha to the 3' end of Pseudomonas exotoxin encoding domains II and III. The resulting protein, PE40-TGF-alpha, accumulated within E. coli and could be purified from extracts of E. coli.
• a cDNA encoding TGF-alpha is placed at the 5' end of a gene encoding domains II and III to produce a TGF-alpha-PE40 fusion protein. This molecule is about 10-fold more active in killing cells bearing EGF receptors than PE40-TGF-alpha.
• TGF-alpha-PE40 In the absence of an ompA signal sequence, TGF-alpha-PE40 was found mainly within the cell but about 25% was found in the periplasm and a small but significant amount was found in the medium. The addition of the ompA signal sequence resulted in large amounts of TGF-alpha-PE40 appearing in the medium as well as in the periplasm. The TGF-alpha-PE40 present in the medium and periplasm is slightly smaller than the TGF-alpha-PE40 molecules found within the cell indicating that processing has taken place as shown in Figure 2 G.
• sequences found within domain II of Pseudomonas exotoxin promote secretion of TGF-alpha-PE40 into the periplasm with some appearing in the medium and the further addition of a signal sequence, such as that of ompA, results in increased secretion of TGF-alpha-PE40 into the medium.
• a signal sequence such as that of ompA
• Examples 16 through 23 illustrate that PhoA fusions are secreted.
• DNA sequences encoding domain II alone or domains II and III were fused to the phoA structural gene which encodes alkaline phosphatase in E. coli.
• Alkaline phosphatase was chosen because it is secreted into the periplasm only when attached to a signal sequence.
• ompA signal sequence were included in some of the constructions.
• the appearance of the fusion proteins into the periplasm and medium was measured in several ways. Aliquots were subjected to PAGE, transferred to nitrocellulose and the proteins located by immunoblotting using an antibody to Pseudomonas exotoxin. In every case the fusion protein had the expected size as shown, for example, in Figure 2H and I.
• the amount of protein present was estimated from the intensity of the antibody reaction and was confirmed by measurements of ADP-ribosylating or alkaline phosphatase activity. Alkaline phosphatase activity is only exhibited by those proteins secreted out of the cellular interior. E. coli BL21 contains a normal alkaline phosphatase gene that is not expressed because the cells are grown in LB with high phosphate. When alkaline phosphatase was placed at the carboxyl terminus of domains II and II (PE40) or domain II alone as shown in Figure 1D, about 8% of the fusion protein appeared in the medium.
• Example 24 illustrates the effect of ompA signal sequence in the level of expression. This example compares selected data presented in the other examples described above. An ATG codon was placed in front of various Pseudomonas exotoxin molecules as shown in Figure 1 and Table 1 beginning with the T7 expression plasmid. When the plasmids containing an ompA signal sequence were constructed, a DNA -fragment was used containing both the ompA SD region and signal sequence. This was fused to various Pseudomonas exotoxin molecules . These plasmids consistently made more recombinant protein than those that did not as shown in Tables 2, 3, and 4.
• ompA has a more efficient SD sequence and (ii) the translation initiation codons in the ompA signal sequence are enriched in A and T and the newly formed RNA can more readily disasssociate from the DNA template and associate with ribosomes.
• LPP outer membrane lipoprotein

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Zoology (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Wood Science & Technology (AREA)
• Gastroenterology & Hepatology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Toxicology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=22707875

Family Applications (1)

Country Status (4)

Families Citing this family (14)

Citations (1)

• 1989
  • 1989-05-04 AU AU37516/89A patent/AU3751689A/en not_active Abandoned
  • 1989-05-04 EP EP19890906964 patent/EP0422049A4/en not_active Withdrawn
  • 1989-05-04 WO PCT/US1989/001837 patent/WO1989010971A1/en not_active Application Discontinuation
  • 1989-05-04 IL IL90190A patent/IL90190A0/xx unknown

Patent Citations (1)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events