WO2010049807A2 - Procédé d'expression recombinante de polypeptides - Google Patents

Procédé d'expression recombinante de polypeptides Download PDF

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Publication number
WO2010049807A2
WO2010049807A2 PCT/IB2009/007384 IB2009007384W WO2010049807A2 WO 2010049807 A2 WO2010049807 A2 WO 2010049807A2 IB 2009007384 W IB2009007384 W IB 2009007384W WO 2010049807 A2 WO2010049807 A2 WO 2010049807A2
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vector
polypeptide
interest
host cell
inducible promoter
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PCT/IB2009/007384
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WO2010049807A3 (fr
Inventor
Youming Zhang
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Gene Bridges Gmbh
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Priority to CN2009801528939A priority Critical patent/CN102264905A/zh
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Publication of WO2010049807A3 publication Critical patent/WO2010049807A3/fr

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/73—Expression systems using phage (lambda) regulatory sequences

Definitions

  • Polypeptides of interest may be recombinantly expressed in prokaryotic host systems using a variety of expression systems.
  • T7-expression system is widely used in E. coli.
  • Target genes are cloned under the control of the T7 promoter, which is recognised by T7 RNA polymerase but not by E. coli RNA polymerase.
  • T7 RNA polymerase is a phage-originating polymerase which is not endogenously present in E. coli.
  • E. coli host cells have now been developed which express T7 RNA polymerase.
  • One example is an E. coli that harbours a lambda bacteriophage carrying the gene coding for T7 RNA polymerase. 1
  • the gene for T7 RNA polymerase was inserted into the host E. coli chromosome (for example E. coli strain BL21 2 ).
  • the pET Expression System is often used under the control of an inducible promoter.
  • the T7 promoter regulated by the lac operator is currently the most widely used inducible protein expression system.
  • the T7 promoter and the lac operator are located 5' of the polypeptide that is to be expressed. Binding of the lac repressor to the lac operator prevents transcription from the T7 promoter.
  • IPTG which is an analogue of lactose
  • IPTG binds to the lac repressor and displaces it from the lac operator.
  • IPTG which is an analogue of lactose
  • lac/IPTG regulated system can only be used in certain E. coli strains (e.g. in BL21).
  • suitable hosts for recombinant polypeptide expression for example, Photorh ⁇ bdus luminescens, Psendomon ⁇ s aeruginosa, Pseiidomonas fliiorencens, Salmonella choleraesuis, Erwinia carotovora, Agrobacteriiim tiimefaciens and Burkholderia pseiidomalleri. It would be useful to have a highly regulatable system for recombinant polypeptide expression that could also be used in other host cells such as these.
  • AMP(s) antimicrobial peptides
  • Conventional antibiotics have been successfully used for more than a century and are widely used for many applications in humans, animals and plants.
  • antibiotic resistance is a growing problem, there has been an alarming drop in the discovery of new antibiotics. 3 4 Consequently, previous liberal and non-essential applications of antibiotics, such as their use as food additives for domestic animals, are being banned. Additionally, many antibiotics frequently used in agriculture have side-effects upon human consumption. To overcome the problems of conventional antibiotics, a new generation of antibiotics is needed.
  • Innate immunity is the principal defence for almost all living organisms 5 and is a rapid and multifunctional defence against microorganisms.
  • 6 7 The antimicrobial function of most innate immunity is mediated by small cationic peptides with potent activity against Gram-positive and Gram-negative bacteria, fungi, parasites, viruses and in certain cases, even mammalian cells. 9 !0 ⁇ These natural product AMPs are potential successors to conventional antibiotics because their antimicrobial mechanism is fundamentally different. 12 The principal mechanism of rapid killing of microbial pathogens by AMPs is attributed to perforation of the microbial cell membrane. 13 14 Consequently, it is very difficult for organisms to establish resistance.
  • AMPs from mammalian use are those with strong activity against microorganisms but not mammalian cells. Therefore, AMPs from animal origins would be good candidates for replacing conventional antibiotics.
  • Lactoferricin B (LfcinB) derived from bovine lactoferrin in milk 15 16 17 and cecropin Pl 18 19 derived from the pig intestine are two of these AMPs and they may serve as alternative antibiotics for animal treatment.
  • Natural AMPs have broad activity against microorganisms but some, such as LfcinB, require high concentrations to be effective.
  • AMPs in large scale are limited because their preparation can be complicated, time consuming and costly.
  • the production of AMPs by chemical synthesis is too costly for them to be used as alternative antibiotics for the treatment of animals, for example of ungulates, pigs, fish or humans. Therefore, a cost effective method for AMP production on industrial scale is required before they can substitute for antibiotic applications on a large scale.
  • AMPs can penetrate bacterial membranes and cause cell lysis, it has been difficult to produce them directly in bacterial cells. Hence the convenience and cost efficiencies of bacterial production have not been accessible.
  • IPTG Induction in the lac/IPTG regulated system relies on IPTG, which is expensive for industrial use. It is also an object of the invention to provide a system for recombinant expression of a polypeptide that is less expensive than current systems that use IPTG.
  • the invention therefore provides a two vector system for broad host range inducible expression of a polypeptide of interest comprising: i). a first vector comprising:
  • a cloning site positioned 3' of the inducible promoter for insertion of a gene encoding the polypeptide of interest; and ii). a second vector for making a host cell competent for T7 RNA polymerase expression, wherein the vector comprises a gene encoding the T7 RNA polymerase under the control of an inducible promoter.
  • the polypeptide of interest is a polypeptide which is toxic to the prokaryotic host, for example, human bactericidal permeability-increasing protein (BPI) 29 or an AMP.
  • the polypeptide of interest is a protein which is difficult to express at very high levels, for example, which is difficult to express at very high levels in the prokaryotic host using standard recombinant expression methods known in the art.
  • the polypeptide of interest is an AMP.
  • the AMP is GB-LfcinB or GB-Cecropin Pl .
  • the polypeptide of interest in addition to being under the control .of the T7 promoter, is also under the control of a further inducible promoter. This lessens the possible toxic effects of polypeptide expression, particularly when the polypeptide of interest is a protein which is toxic to the prokaryotic host.
  • the DNA encoding the polypeptide of interest is transcribed by two RNA polymerases: the T7 RNA polymerase and the endogenous host RNA polymerase. This is the first system that we are aware of in which a gene is transcribed using two separate RNA polymerases.
  • the inducible promoter will preferably have an active state, in which an RNA polymerase can bind to and transcribe from the promoter, and an inactive state, in which the same RNA polymerase cannot transcribe from the promoter.
  • the inducible promoter is bound by a repressor when the promoter is in its inactive state. Removal of the repressor from the promoter then moves the inducible promoter to its active state. Removal of the repressor may be by way of binding of an inducer to the repressor.
  • the repressor may be endogenously expressed in the host cell. Alternatively, the host cell may be transformed with a gene encoding the repressor. In a preferred embodiment, a gene encoding the repressor for the inducible promoter is present on the first and/or the second vector of the invention.
  • the inducible promoter is preferably a promoter that functions in a broad host range. Tetracycline inducible promoters such as Ptet 20 are particularly preferred. Use of tetracycline as the inducer, instead of IPTG, enables the system to be used in a broad host range and also results in a cheaper system because tetracycline is less expensive than IPTG. Indeed, currently, tetracycline or its derivatives (for example, anhydride- tetracycline and doxycycline) are more than 100 times cheaper than IPTG. In addition, the amount of tetracycline used for induction is more than 100 times less than the amount of IPTG used for induction.
  • the total cost for tetracycline is at least 10 4 times less than for IPTG.
  • any other suitable inducible promoter may be used, for example, the doxycycline-regulated T7 promoter or sugar inducible promoters such as the lac operator, the BAD promoter (arabinose inducible) or the Rhamnose inducible promoter.
  • sugar inducible promoters pose two problems in that 1) they do not work in a broad range of bacterial host strains as does the tetracycline inducible promoter, and 2) the inducer is more expensive than tetracycline.
  • the skilled person will be able to determine the amount of the inducer, such as tetracycline or its derivatives, to be used for a particular host cell.
  • the host cell is a prokaryotic cell.
  • Tetracycline or its derivatives are toxic to some prokaryotic cells when used at high doses such as 3 micrograms per ml of bacterial culture.
  • Different prokaryotic strains have different resistance properties to tetracycline and its derivatives.
  • the present invention therefore utilises tetracycline or its derivatives at a dose that is lower than the minimal dose required for toxicity to the host cell.
  • Preferred amounts of tetracycline or its derivatives are 0.5-1 microgram per ml of bacterial culture.
  • a cloning site is positioned 3' of the inducible promoter for insertion of a gene encoding the polypeptide of interest.
  • the gene encoding the polypeptide of interest may be inserted into the cloning site by any suitable method.
  • the cloning site comprises two restriction sites for cloning of the gene encoding the polypeptide of interest. These two restriction sites may be any suitable restriction sites.
  • the use of a restriction site at the 5' end that maintains the ribosomal binding site distance and the AT dinucleotides in front of the ATG of the gene of interest is preferred.
  • EcoRV is a particularly preferred restriction site for use at the 5' end.
  • the presence of AT dinucleotides in front of ATG is particularly preferred for peptide expression in E. coli.
  • An example of a suitable restriction site that may be used at the 3 ' end is PvuII.
  • the cloning site comprises a restriction site which can be used for linearizing the vector.
  • a linear DNA fragment carrying the gene encoding the polypeptide of interest with sequences homologous to the vector flanking the gene may then be combined with the linearized vector to form the vector using homologous recombination, for example, using recombineering techniques.
  • the cloning site comprises two regions of homology with a linear DNA fragment carrying the gene encoding the polypeptide of interest and the linear DNA fragment is inserted into the vector by homologous recombination, for example, using recombineering techniques.
  • Recombineering techniques are known in the art, for example, see WO 99/29837, WO 01/04288, WO 02/062988 and Wang J et al. 2006, MoI Biotechnol. 32(l):43-53; Muyrers, J.P.P. et al. 2001, Trends in Biochemical Sciences, 26, 325-31.).
  • the cloning site comprises one or more restriction sites
  • these one or more, restriction sites are preferably not found elsewhere in the vector.
  • the first vector comprises the polypeptide of interest at the cloning site.
  • the first vector comprises the polypeptide of interest between the two restriction sites.
  • the first vector advantageously comprises a terminator sequence positioned 3 ' of the cloning site.
  • the presence of the terminator sequence 3 ' of the cloning site ensures that, when transcription occurs from the T7 promoter, it stops at the terminator sequence and does not run past this sequence.
  • the presence of terminator sequences results in an increase in protein expression efficiency compared to when the terminator sequence is not present because non-stop transcription does not occur.
  • the terminator is the T7 terminator.
  • the sequence of the T7 terminator sequence is shown in Figure 2.
  • the first vector comprises the cassette (also known as a construct) T7 promoter-inducible promoter-cloning site-terminator. More preferably, the first vector comprises the cassette T7 promoter-Ptet-EcoRV-[3' restriction site]-T7 terminator. Even more preferably, these vectors comprise the gene encoding the polypeptide of interest inserted at the cloning site. These vectors also preferably comprise a gene encoding the repressor for the inducible promoter, such as the tetR repressor.
  • the first vector comprises the sequence shown in Figure 2 or Figure 3. More preferably, the first vector is a plasmid as shown in Figure 2 or Figure 3.
  • the second vector is used to ensure that the host cell is competent for T7 RNA polymerase expression.
  • the gene encoding T7 RNA polymerase comprises or consists of the sequence provided in GenBank No. M38308, more preferably as shown in Figure 7, or encodes a T7 RNA polymerase having the sequence provided in M38308, more preferably as shown in Figure 8.
  • the gene encoding T7 RNA polymerase is a variant of the sequence shown in M38308 or Figure 7, for example, it has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence shown in M38308 or Figure 7, and encodes a functional T7 RNA polymerase.
  • the gene encoding T7 RNA polymerase encodes a fragment or a variant of the T7 RNA polymerase of M38308 or Figure 8.
  • the gene encoding T7 RNA polymerase may be a fragment of the sequence shown in M38308 or Figure 7 or a variant thereof, wherein the fragment encodes a functional T7 RNA polymerase.
  • the fragment may be at least 2100 nucleotides in length, at least 2400 nucleotides in length, at least 2700 nucleotides in length, at least 2772 nucleotides in length, at least 2800 nucleotides in length, at least 2812 nucleotides in length, or at least 2818 nucleotides in length.
  • the gene encoding the T7 RNA polymerase is under the control of an inducible promoter. This ensures that the gene encoding the T7 RNA polymerase is transcribed and subsequently expressed only when the inducible promoter is in its active state and advantageously results in an additional level of regulation compared to systems in which T7 RNA polymerase is constitutively expressed.
  • the inducible promoter on the second vector is the same as the inducible promoter that is present on the first vector. In other embodiments, the inducible promoter on the second vector is different from the inducible promoter that is present on the first vector. Having both the gene encoding T7 RNA polymerase and the gene encoding the polypeptide of interest under control of the same inducible promoter advantageously ensures that expression of the polypeptide of interest is highly regulated by the particular state of the inducible promoter. Use of the same inducible promoter on both vectors also results in rapid transcription and subsequent expression of the polypeptide of interest when the inducible promoter is in the active state.
  • the inducer of the inducible promoter in both vectors is tetracycline or a derivative thereof.
  • each vector may comprise an origin of replication which is functional in the host cell or the cassettes may be maintained in the host cell by integration into the host chromosome or into an endogenous mobile vector.
  • the part of the first vector which comprises the cassette T7 promoter- inducible promoter-cloning site may be maintained in the host via a different mechanism from the part of the second vector which comprises the gene encoding the T7 RNA polymerase under the control of an inducible promoter.
  • the origin of replication in the first vector may be the same as or different from the origin of replication in the second vector.
  • the origin of replication may be any suitable origin such as a plasmid origin or a phage origin.
  • the origin of replication is functional in the host cell.
  • the first vector comprises an origin of replication that is functional in the host cell, such as the colEl ori.
  • the second vector comprises an origin of replication that is functional in the host cell and so the vector is maintained in the host cell and T7 RNA polymerase is expressed from the vector.
  • the origin of replication in the second vector is non-functional in the host cell that is being made competent for T7 RNA polymerase expression. This ensures that the second vector does not replicate in the host cell which the vector is destined to make competent for T7 RNA polymerase expression so that maintenance of the region present between the two inverted repeat regions ("IR(s)") which comprises the gene encoding T7 RNA polymerase requires integration of the region in the host chromosome.
  • IR(s) inverted repeat regions
  • Any suitable origin of replication which is non-functional in the host cell may be used.
  • the origin is preferably functional in other cells such that the vector may be prepared and/or the vector may be replicated and maintained prior to use in the present invention.
  • the origin of replication in the second vector is the R6K origin of replication.
  • the R6K origin of replication requires the pir protein 21 to replicate and so the second vector may be constructed and maintained in another cell that expresses the pir protein prior to use in the host cell of the invention.
  • a vector having ori R6K as the only origin of replication will not replicate in cells that do not express the pir protein.
  • Any other suitable origin of replication may be used in the second vector, for example, RK2. 22
  • the origin of replication in the second vector is situation between the inverted repeats in the region which comprises the gene encoding the T7 RNA polymerase. It is sometimes desirable to determine the position of integration of the cassette in the host chromosome. A simple way of doing this would be to prepare genomic DNA, digest with one restriction enzyme, and then ligate and transform into a pir strain to rescue the R6K ori containing vector. The vector would now contain part of the genomic DNA where it was integrated. By sequencing the junction, one can see the integration position in the chromosome. Thus, the presence of the origin of replication between the inverted repeats is advantageous because it makes it easier to detect the insertion position by plasmid rescue.
  • the gene encoding the T7 RNA polymerase is preferably situated between two inverted repeat regions that are recognised by a transposase such that the sequence between the two inverted repeats is integrated into the chromosome by the transposase.
  • Any suitable inverted repeat regions that are recognised by a transposase may be used.
  • suitable inverted repeat regions that are recognised by the MycoMar transposase are 5' ACAGGTTGGCTGATAAGTCCCCGGTCT ... AGACCGGGGACTTATCAGCCAACCTGT 3'.
  • transposase is transient such that once the inverted repeat cassette region has integrated into the host chromosome, the transposase is no longer expressed.
  • the transposase is encoded by the second or first vector.
  • the gene encoding the transposase is present in the second vector but is not present in the region between the two inverted repeats which contains the gene encoding the T7 RNA polymerase. This ensures that the gene encoding the transposase is not integrated into the host cell chromosome. Any suitable transposase may be used.
  • the transposase is the MycoMar transposase. 23
  • the first and/or second vector advantageously comprises a selection gene.
  • Both vectors preferably comprise a selection gene.
  • the selection gene on the first vector is preferably different from the selection gene on the second vector.
  • the selection gene in the second vector is preferably situated between the two inverted repeats, where present, in the region that comprises the gene encoding the T7 RNA polymerase so that host cells which have integrated this region into their chromosome can be selected for.
  • Any suitable selection genes may be used, for example, antibiotic resistance genes such as the genes for gentamycin resistance, neomycin resistance, ampicillin resistance or hygromycin resistance, or host complementary genes.
  • the selection gene is preferably removable, particularly where the cassette integrates into the host chromosome.
  • the selection gene may be flanked by sites that can be used to excise the selection gene.
  • the sites that can be used to excise the selection gene are loxP sites, for example, mutated loxP sites (loxP*), preferably Iox66 and 71.
  • Cre recombination can be used to remove the selection gene after integration.
  • the second vector comprises a removal selection gene which is flanked by mutated loxP sites and wherein the selection gene is located between the inverted repeats in the region which comprises the gene encoding the T7 RNA polymerase.
  • the second vector comprises the cassette IR- [origin of replication]-loxP*-[selection gene]-loxP*-tetR-Ptet-T7RNApol-IR.
  • This vector preferably also comprises a gene encoding a transposase wherein the transposase is not present in the region between the two inverted repeats which contains the gene encoding the T7 RNA polymerase.
  • the second vector comprises the cassette IR-oriR6K-loxP*-genta-loxP*-tetR-Ptet-T7RNApol-IR.
  • the second vector is a plasmid as shown in Figure 1.
  • the T7 promoter is positioned immediately 5' of the inducible promoter such that there is no intervening sequence between them.
  • the two restriction sites are located such that transcription of the gene encoding the polypeptide of interest may occur from the T7 promoter and from pTet.
  • the cloning site (for example, the two restriction sites) is positioned immediately 3 ' of the inducible promoter such that there is no intervening sequence between them.
  • the T7 terminator sequence is positioned immediately 3' of the cloning site such that there is no intervening sequence between them.
  • intervening sequence is present between the T7 promoter and the inducible promoter and/or between the cloning site and the inducible promoter and/or between the T7 terminator sequence and the cloning site.
  • the intervening sequence is preferably less than 400bp in length, less than 250bp in length, less than lOObp in length, less than 50bp in length, less than 20bp in length, less than 5bp in length or less than 3bp in length.
  • the sequences of the T7 promoter and the T7 terminator are shown in Figures 2, 3A and 3B and in the descriptions thereof.
  • the vectors for use in the invention are any suitable vectors that may be used to transform a host cell, preferably a prokaryotic host cell.
  • the first and/or second vector is a plasmid vector.
  • the first and/or second vector is ,-a phage vector.
  • the first vector is a plasmicL vector and the second vector is a phage vector.
  • the first vector is a phage vector and -the second vector is a plasmid vector.
  • the polypeptide of interest may be part of a fusion protein.
  • Particularly preferred fusion proteins are the fusion of the polypeptide of interest to an inclusion body protein or to a crystal-forming inclusion body protein.
  • a crystal-forming inclusion body protein aggregates in the form of a regular structure.
  • the incorporation of the polypeptide of interest into inclusion bodies permits expression of the polypeptide of interest whilst masking any potential toxic effects of the polypeptide to the host cell.
  • the polypeptide of interest has antibiotic activity
  • fusion with the inclusion body protein masks the antibiotic activity.
  • the polypeptide of interest is toxic to the host cell, for example, if it is an AMP.
  • the system provided by the present invention allows very high levels of expression of the polypeptide of interest to be obtained.
  • the system of the invention may be used to manufacture AMPs at an industrial scale.
  • inclusion body proteins suitable for use in the invention are crystal proteins from Photorhabdus himinescens, Xenorhabdiis nematophilus and Bacillus thuringiensis. 25
  • Crystal-forming inclusion body proteins are particularly preferred inclusion body protein fusion partners. Any suitable crystal-forming inclusion body protein may be used. Those obtainable from Photorhabdus or Xenorhabdiis are good examples as they are expressed at very high levels. In addition, they naturally aggregate to form insoluble bodies and will be very easy to purify via harvest of their inclusion bodies. In a preferred embodiment, the crystal-forming inclusion body protein is Photorhabdus CipB. This small protein of 100 amino acids can accumulate in cells to up to 40% of total protein. 27 The inventors have found that, using the two vector system of the present invention, very high levels of expression of the polypeptide of interest are obtained when the polypeptide is fused to CipB.
  • up to 70% of the total protein includes the polypeptide.
  • any other suitable crystal-forming inclusion body may be used, such as Photorhabdus CipA 27 or Xenorhabdiis nematophila PixA 28 .
  • Fragments and variants of an inclusion body protein may also be used wherein the fragment or variant retains the function of the full length or native sequence protein insofar as it forms an inclusion body.
  • the inclusion body protein is preferably fused 5' of the polypeptide of interest.
  • the inclusion body protein is fused to the polypeptide of interest via a cleavable linker which permits rapid and simple release of the polypeptide of interest from the inclusion body protein.
  • the cleavable linker is an Asp-Pro dipeptide linker between the inclusion body protein and the N-terminus of the polypeptide of interest.
  • the Asp-Pro dipeptide linker may be cleaved using HCl 29 . In other embodiments, any other suitable cleavable linker may be used.
  • the polypeptide of interest has toxic activity
  • the polypeptide of interest is toxic to the host cell (for example, where it is an AMP)
  • the incorporation of the polypeptide of interest into inclusion bodies permits very high levels of expression without toxic activity and also allows rapid and simple purification.
  • the first vector preferably comprises the cassette: 5'-T7 promoter-inducible promoter-gene encoding the inclusion body protein fused 5' of the gene encoding the polypeptide of interest-terminator-3 '.
  • the use of the system of the present invention also advantageously allows expression of AMPs in industrially convenient prokaryotic hosts without interfering with the growth of the hosts.
  • the invention therefore provides a polynucleotide encoding a fusion protein comprising a crystal-forming inclusion body protein fused to a polypeptide of interest.
  • the polypeptide of interest is less than 500 amino acids in length, for example, less than 200 amino acids in length, less than 150 amino acids, less than 100 amino acids, less than 80 amino acids, less than 60 amino acids, less than 50 amino acids, less than 30 amino acids or less than 20 amino acids in length.
  • the polypeptide of interest is an AMP.
  • AMPs that may be used are lactoferricin B or cecropin Pl.
  • the invention provides AMPs that have an additional proline at their 5' end compared to the natural sequence of the AMJk. The presence of the proline is an artefact of cleavage of the Asp-Pro dipeptide linker in the fusion- protein. The inventors have found that, surprisingly, the presence of this 5' proline does not affect the antibiotic activity of the AMP.
  • a particular example of a polypeptide of interest that may be used in and is provided by the invention is the GB-LfcinB derivative of lactoferrin having the sequence PAWFKCWRWQWRWKKLGA.
  • Also provided by the invention is a polypeptide of interest fused to a crystal forming inclusion body protein, as described above.
  • the invention further provides a method for expressing a polypeptide of interest which comprises:
  • the T7 RNA polymerase is under the control of an inducible promoter which is the same as the inducible promoter in the first vector.
  • the host cell may already comprise the T7 RNA polymerase.
  • it may be necessary to provide the cell with the T7 RNA polymerase and so the method for expressing a polypeptide of interest may comprise:
  • the first and the second vector may be transformed into the host cell simultaneously or in any order.
  • the first vector may be transformed into the host before the second vector.
  • the second vector may be transformed into the host before the first vector.
  • the host cell After transforming the host cell, the host cell is preferably grown to a suitable cell density before adding the inducer of the inducible promoter.
  • a suitable cell density is .one at which there are enough cells present to express the polypeptide ⁇ interest at the desired level. In some embodiments, a suitable cell density is between OD600 -0.4 to OD600 -2.0, for example OD600 -0.7.
  • the cells are preferably grown to a final concentration of between 0.1 mg/ml to 1.5 mg/ml, more preferably about 0.5 mg/ml, before the inducer (preferably tetracycline) is added.
  • the cells may then harvested after a suitable period of time after transformation with the first (and, in some embodiments, second) vectors to allow for production of the polypeptide of interest, for example, between 12 to 20 hours, more preferably about 16 hours.
  • the method may additionally comprise the steps of purifying inclusion bodies comprising the fusion protein and cleaving the cleavable linker between the inclusion body protein and the polypeptide of interest to release the polypeptide of interest.
  • the cleavable linker is the Asp-Pro dipeptide linker
  • the method may comprise adding HCl to the host cell to cleave the linker. The cleavage step is preferably carried out after harvest of the cells.
  • HCl is added to the cells at a suitable concentration to cleave the linker.
  • the addition of HCl also serves to lyse the cells which results in cleavage of the polypeptide of interest from the inclusion body protein and liberation of the polypeptide of interest from the host cell in one single step.
  • 0.05-0.2 N HCl is used to lyse the cells.
  • the use of 0.1N HCl is preferred.
  • the incubation period with HCl varies depending upon the concentration of HCl being used. The incubation time to be used will be clear to the skilled person.
  • the cells are preferably incubated with the HCl (preferably with 0.1N HCl) at high temperature for a period of time sufficient to cleave the linker and lyse the cells.
  • the cells are incubated with the HCl at a temperature of between 7O 0 C and 95 0 C for about 30 mins to 4 hours. An incubation time of about 2 hours at about 85 0 C is preferred.
  • the method may further comprise a chromato graph procedure to further purify the polypeptide of interest following its release from the host cell.
  • the host cell may be prokaryotic or eukaryotic. Preferably the host cell is prokaryotic.
  • the host cell preferably does not express the pir protein in embodiments in which the origin of replication in the second vector is R6K...
  • the host cell is selected from E. coli Kl 2, E. coli Nissle 1917, Photorhabdus, Pseudomonas, Agrohacteriwn, Myxococcus and Burkholderia.
  • the host cell may be Photorhabdus luminescens, Pseudomonas aeruginosa, Pseudomonas fluorencens, Agrobacterium tumefaciens or Burkholderia pseudomalleri.
  • the host cell is Salmonella choleraesuis or Erwinia carotovora. Li other embodiments, the host cell is »5. cerevisiae.
  • kits comprising a first vector according to the invention and a second vector according to the invention.
  • the polypeptide of interest is preferably a polypeptide that is toxic to the prokaryotic host, such as an AMP.
  • the system of the present invention advantageously allows AMPs to be produced in large quantities so that they can be used for industrial applications.
  • AMPs a polypeptide that is toxic to the prokaryotic host
  • the system of the present invention advantageously allows AMPs to be produced in large quantities so that they can be used for industrial applications.
  • Recently, the use of conventional antibiotics to treat animals has been banned by the European Union. Consequently, animal loss due to bacterial infection is once again a major problem for farmers.
  • the major problem for the piglets is diarrhoea from infection caused by microorganisms. This period is the most dangerous time and the conventional way to treat the piglet is to give antibiotics for a few days.
  • the method of the invention allows for the first time, the large scale production of AMPs which enables the large scale use of AMPs in agriculture and aquaculture, which was not possible prior to the present • invention.
  • the present invention therefore provides a method for treating or preventing bacterial infection in animals comprising use of an AMP produced by the system of the invention.
  • the animals are humans or other mammals such as baby animals, for example piglets or calves, particularly those baby animals that have been taken away from their mothers.
  • the invention provides a method for treating or preventing bacterial infections in piglets comprising administering GB-LfcinB or GB-Cecropin Pl .
  • the piglets are those piglets who have been removed from their mother within the past 10 days, more preferably within the past 5 days, more preferably within the past 1 or 2 days or, for example, who are to be removed from their mother within the next 1-10 days.
  • the bacterial infection is preferably E. coli infection.
  • Use of the AMP preferably prevents diarrhoea in the piglet and/or results in the piglet gaining body weight.
  • lmg of either GB-LfcinB or GB-Cecropin Pl is administered to a piglet in a single dose.
  • 0.5-2mg or 2-3mg may be administered in a single dose.
  • 2mg or more than 2mg are given as a single dose, for example 3-5mg, 5-8mg or 8-1 Omg is given as a single dose.
  • only a single dose is given.
  • multiple doses are given to each piglet, for example, on consecutive days or on every other day.
  • the animals are fish.
  • a method for treating or preventing bacterial infection in fish comprising adding AMPs produced by a method of the invention to the water in which the fish are swimming.
  • the treating or preventing bacterial infection in fish comprises adding the AMPs in a quantity of at least O.lmg, at least 0.5mg, at least lmg, at least 2mg, at least 5mg, at least 500mg, at least Ig, at least 5g, at least 1Og, at least 5Og, at least 10Og, at least 300g, at least 50Og, at least 75Og, at least lkg, at least 2kg to the water in which the fish are swimming.
  • adding the AMP to water achieves a concentration of at least O.OOlmg AMP per litre, at least O.Olmg per litre, at least O.lmg per litre, at least lmg per litre, at least lOmg per litre, at least lOOmg per litre, at least 500mg per litre, or at least Ig per litre.
  • the ability to manufacture large quantities of the AMPs is particularly advantageous in embodiments in which the water in which the fish are present is part of a lake, pond, stream, sea, ocean, or other large body of water. rHowever, the method is also applicable to treating or preventing bacterial infection in fish present in other bodies of water such as in a bowl or tank.
  • the skilled person will be able to determine how much AMP to add depending on the volume of water that is present, the potential for dispersal of the AMP in that volume of water, the potency of the particular AMP and the number offish that are present.
  • AMPs also have the potential to act as food preservatives and so the invention provides the use of an AMP produced by a method of the invention as a food preservative.
  • the AMP is added to food in order to achieve a concentration of at least O.OOOlmg AMP per cm 3 of food, at least O.OOlmg AMP per cm 3 of food, at least O.Olmg AMP per cm 3 of food, at least O.lmg AMP per cm 3 of food, at least lmg AMP per cm 3 of food, or at least 5mg AMP per cm 3 of food.
  • AMPs of the invention for use in any of these methods.
  • Figure 1 Universal T7 RNA polymerase expression system
  • FIG. 1 Broad host range inducible expression vector; the sequence of the T7 promoter (TAATACGACTCACTATA) , the Ptet promoter
  • FIGS 3 A and 3B Figures 3 A and 3B.
  • GB-Cecropin Pl as an example for AMP expression in microorganisms.
  • Figure 4 Expression of AMPs in E. coli with the oriR6K-loxP*-genta-loxP*-tefR- Ptet-T7RNApol cassette in the chromosome;
  • FIG. 6 Body-weight of piglets after challenge with E. coli. The bodyweight of piglets slaughtered without any treatment was used as the baseline (0).
  • Tag 1-5 i.e. "Day” 1-5) means the bodyweight measured at day 33 (5 days after challenge and AMPs) and
  • Tag 6-11 means the bodyweight measured at day 39 (11 days after challenge and AMPs).
  • Tag 1-11 represents the average bodyweight increasing;
  • FIG. 7 T7 RNA polymerase nucleotide sequence (coding sequence and ribosomal binding site).
  • FIG. 8 T7 RNA polymerase amino acid sequence.
  • the first vector expresses T7 polymerase from a tetracycline regulated promoter.
  • This vector is a plasmid is based on the R6K origin and can only replicate in the presence of the pir protein.
  • the IR- oriR6K-loxP*-genta-loxP*-tetR-Ptet-T7RNApol-IR cassette will be integrated into the chromosome because of transposition mediated by TPase (MycoMar Transposase) acting on the inverted repeats (IRs).
  • TPase MycoMar Transposase
  • IRs inverted repeats
  • the IR (inverted repeat) - IR cassette is used for chromosomal integration by transposition mediated by the encoded transposase (Tpase, which is not integrated). Between the IRs, in addition to the T7 gene, the following features are present: the oi ⁇ R6K, which requires pir protein to replicate; the gentamycin resistance gene, which is used for integration selection and is flanked by mutated loxP* sites (Iox66 and 71), which can be used to excise the gentamycin resistance gene, so that gentamycin can be used for host selection again; and the gene for the tetracycline repressor (tetR). Although the example is exemplified with the use of the gentamycin resistance gene, any selection gene can be used in this vector.
  • E. coli Kl 2 E. coli Nissle 1917, Photorhabdus, Pseudomonas, Agrobacterium, Myxococcus and Burkholderia have been transformed by this vector.
  • T7 RNA polymerase can be expressed in these transformants after tetracycline induction.
  • the second vector is a plasmid carrying the gene of interest (i.e. the CipB-AMP fusion; Figure 3).
  • This gene is cloned under a dual, T7 and Ptet double promoter, which is doubly regulated by tetracycline, once via tetracycline induction of the T7 polymerase and once directly via derepression of the tetracycline repressor (TetR).
  • TetR tetracycline repressor
  • the gene of interest is silent because the T7 RNA polymerase gene and Ptet are repressed by tetR. After adding tetracycline, both promoters are derepressed and the T7 promoter is activated by T7 polymerase, resulting in massive induction.
  • T7 RNA polymerase is under a more regulated promoter - tetracycline inducible promoter;
  • the tetracycline inducible promoter is a broad- range promoter and can be used in many bacterial strains;
  • the inducer, tetracycline is more than 10 4 times less costly than IPTG so can be applied at industrial scale.
  • Example 2 Structure and expression of the fusion proteins
  • FIG. 3 shows one such example after cloning of a CipB-AMP (here GB-Cecropin Pl) fusion protein gene.
  • CipB-AMP here GB-Cecropin Pl
  • the cells were directly dissolved in 0.1N HCl for digestion.
  • Figure 5 shows that AMPs can be released from CipB after 2 hours treatment of 0.1N HCl at 85°C.
  • Bacterial cells in 0.1N HCL were incubated at 85°C for 2 hours. Samples were taken out at 0.5 hours, 1.0 hour and 2 hours for SDS-PAGE checking. Samples 1, 2 and 3 were from lanes 2, 4 and 6 in Figure 4.
  • Sample 1 is GB-Cecropin Pl
  • sample 2 is GB-Buforin II
  • sample 3 is GB-LfcinB. Released AMPs can be further purified by a chromatograph procedure.
  • Example 3 Challenge experiment of piglets
  • GB-Cecropin Pl GB-Ce Pl
  • GB-Lactoferricin B LfcinB 15
  • Cecropin 18 ' 19 is a polypeptide composed of 31 amino acids and its sequence is SW LSK TAK KLE NSA KKR ISE GIA IAI QGG PR (Ser-Trp-Leu-Ser-Lys-Thr-Ala- Lys-Lys-Leu-Glu-Asn-Ser-Ala-Lys-Lys-Arg-Ile-Ser-Glu-Gly-Ile-Ala-Ile-Ala-Ile-Gm- Gly-Gly-Pro-Arg).
  • GB-Ce Pl is cecropin Pl plus a proline and has the sequence PSW LSK TAK KLE NSA KKR ISE GIA IAI QGG PR
  • Lactoferricin B (LfcinB) is isolated by pepsin digestion of bovine lactoferrin (Lf).
  • LfcinB is the 25 amino acid sequence from amino acid 17 to amino acid 41 of bovine Lf.
  • LfcinB has the sequence
  • FKCRRWQWRMKKLGAPSITCVRRAF Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg- Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe. More recently, the first 15 amino acids of LfcinB (FKCRRWQ WRMKKLGA) was found to be the active region. Although the milk from other organisms contains similar LfcinB peptides, the one from bovine (LfcinB) has the strongest activity against bacteria, fungi and even viruses.
  • LfcinB has moderate activity against the microorganisms (MIC between 12-100 ⁇ g/ml). By changing some amino acids in LfcinB, other LfcinB derivatives have better activity than the original LfcinB peptide 32 .
  • GB-LfcinB is the peptide generated by exchanging the amino acids according to the activity prediction. 33 A proline at the first position arises from the biosynthesis procedure. Thus, the sequence of GB-LfcinB is PA WFKCWRWQ WRWKKLGA.
  • GB-Cecropin Pl and GB-LfcinB 1 were used for animal experiments using the experimental protocol described in Table 1.
  • Group 0 (12 piglets) were slaughtered without any treatment at day 28 as a control.
  • Group 1 was challenged two times at day 28 and day 29 with a pig pathogenic E. coli strain which can cause diarrhea in piglets.
  • Group 2 was challenged with pathogenic E. coli at day 28 and lmg of GB-LfcinB was given to piglets after pathogenic E. coli challenge.
  • the piglets were challenged once again with pathogenic E. coli.
  • Group 3 was treated the same as Group 2 but with a different AMP (GB-Ce Pl). 6 piglets were slaughtered at day 33 from Groups 1-3 and another 6 were slaughtered at day 39. Diarrhea and bodyweight of the piglets were checked after they were slaughtered. Diarrhea appeared in challenged piglets (Table 2). However, the data show that less than half of the challenged piglets got diarrhea after treatment with AMPs.
  • Bodyweight of each slaughtered piglet was measured at day 28, day 33 and day 39.
  • the data in Figure 6 show the bodyweight gained or lost after challenge " with E. coli at day 33 and day 39. It shows that E. coli-alone challenged piglets (Group 1) lost around 0.08kg bodyweight after 5 days. With additional AMP GB-LfcinB, piglets lost around 0.05kg (Group 2). With GB-Ce Pl, the piglets (Group 3) only lost 0.005kg bodyweight. At average after 11 days, the piglets treated with GB-Ce Pl gained two times more bodyweight than the control group.

Abstract

L’invention concerne un système de deux vecteurs permettant l’expression, pouvant être induite par un large spectre d’hôtes, d’un polypeptide d’intérêt. Ledit système comprend : i) un premier vecteur comprenant : (a) le promoteur T7 positionné en 5’ d’un promoteur inductible; (b) un site de clonage positionné en 3’ du promoteur inductible pour introduire un gène codant pour le polypeptide d’intérêt; et ii) un second vecteur qui rend une cellule hôte compétente pour exprimer l’ARN polymérase T7, ledit vecteur comprenant un gène codant pour l’ARN polymérase T7 sous la commande d’un promoteur inductible.
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EP2628793A1 (fr) * 2012-02-17 2013-08-21 Pharma-Zentrale Gmbh Souches de Escherichia Coli recombinantes
WO2016180379A1 (fr) * 2015-05-14 2016-11-17 Universidad De Los Andes Nouveau transposon favorisant l'expression fonctionnelle de gènes dans des adn épisomaux et procédé d'augmentation de la transcription d'adn dans des analyses fonctionnelles de bibliothèques métagénomiques
CN107212155A (zh) * 2017-06-16 2017-09-29 西南大学 一种用于鲤科家鱼养殖的药物组合物
EP3330282A1 (fr) * 2016-12-02 2018-06-06 Ludwig-Maximilians-Universität München Cipa et cipb pixa comme échafaudages pour organiser des protéines dans des inclusions cristallines
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CN115197954A (zh) * 2021-04-14 2022-10-18 上海凯赛生物技术股份有限公司 用于发酵生产1,5-戊二胺的重组dna、菌株及其用途
CN116769814B (zh) * 2023-06-16 2024-02-02 苏州泓迅生物科技股份有限公司 一种大肠杆菌益生菌t7表达系统及其应用

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EP2628793A1 (fr) * 2012-02-17 2013-08-21 Pharma-Zentrale Gmbh Souches de Escherichia Coli recombinantes
WO2013120618A1 (fr) * 2012-02-17 2013-08-22 Pharma-Zentrale Gmbh Souches recombinantes d'escherichia coli
CN103145848A (zh) * 2013-02-06 2013-06-12 上海高龙生物科技有限公司 重组乳铁蛋白二肽、表达载体及其应用
CN103145848B (zh) * 2013-02-06 2014-11-26 上海高龙生物科技有限公司 重组乳铁蛋白二肽、表达载体及其应用
WO2016180379A1 (fr) * 2015-05-14 2016-11-17 Universidad De Los Andes Nouveau transposon favorisant l'expression fonctionnelle de gènes dans des adn épisomaux et procédé d'augmentation de la transcription d'adn dans des analyses fonctionnelles de bibliothèques métagénomiques
US11155822B2 (en) 2015-05-14 2021-10-26 Universidad De Los Andes Transposon that promotes functional DNA expression in episomal DNAs and method to enhance DNA transcription during functional analysis of metagenomic libraries
EP3330282A1 (fr) * 2016-12-02 2018-06-06 Ludwig-Maximilians-Universität München Cipa et cipb pixa comme échafaudages pour organiser des protéines dans des inclusions cristallines
WO2018172739A1 (fr) * 2017-03-24 2018-09-27 Fujifilm Diosynth Biotechnologies Uk Limited Système d'expression
CN107212155A (zh) * 2017-06-16 2017-09-29 西南大学 一种用于鲤科家鱼养殖的药物组合物
CN107212155B (zh) * 2017-06-16 2019-12-17 西南大学 一种用于鲤科家鱼养殖的药物组合物

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