WO1991001374A1 - Prevention d'un commencement de translation interne - Google Patents

Prevention d'un commencement de translation interne Download PDF

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Publication number
WO1991001374A1
WO1991001374A1 PCT/US1990/004113 US9004113W WO9101374A1 WO 1991001374 A1 WO1991001374 A1 WO 1991001374A1 US 9004113 W US9004113 W US 9004113W WO 9101374 A1 WO9101374 A1 WO 9101374A1
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WO
WIPO (PCT)
Prior art keywords
codon
shine
sequence
internal
initiation
Prior art date
Application number
PCT/US1990/004113
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English (en)
Inventor
Frank Genbauffe
Original Assignee
Seragen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seragen, Inc. filed Critical Seragen, Inc.
Publication of WO1991001374A1 publication Critical patent/WO1991001374A1/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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • This invention relates to the use of recombinant DNA techniques to maximize the expression and purity of useful polypeptides.
  • a critical stage in the expression of a protein is the initiation of translation. Initiation is the point at which a ribosome, a mRNA molecule, a charged tRNA, and other factors are assembled in the proper relationship to allow the insertion of the initial amino acid residue of the polypeptide to be synthesized.
  • prokaryotes In prokaryotes the initiation of translation is controlled in large part by signals encoded in the sequence of mRNA molecules. Initiation of prokaryotic translation begins with the binding of a 3OS ribosomal subunit to an mRNA molecule. A ribosomal binding site usually precedes the initiator codon of each independently synthesized polypeptide product. Thus a polycistronic prokaryotic message encoding three polypeptides will generally possess a ribosome binding site just 5' to each of its independently synthesized polypeptide products.
  • Shine- Dalgarno sequence Only the core of the ribosomal binding site, the Shine- Dalgarno sequence, is conserved between various bacterial genes, and even the Shine-Dalgarno sequence is not invariant.
  • a consensus Shine-Dalgarno sequence is AGGAGG.
  • the Shine-Dalgarno sequence is complimentary to a pyrimidine-rich portion of the 16S ribosomal RNA component of the 3OS ribosomal subunit.
  • the Shine-Dalgarno sequence is generally centered approximately 4 to 20 nucleotides upstream from an initiator codon.
  • the initiator codon in bacteria is generally AUG, but may be GUG or even TJUG or ATJU.
  • the amino terminal amino acid, the first added in the synthesis of the polypeptide, is almost invariably N-formylmethionine (fMet) .
  • Formylation occurs after the methionine has been attached to its tRNA.
  • the formyl group blocks the amino group of the methionine. This does not prevent the use of N-formylmethionine at the first position in the polypeptide chain but precludes its insertion at any other point,because of the inability of the blocked amino group to participate in a peptide bond.
  • the formyl group is usually removed from the amino terminal methionine by a deformylase. In the case of many proteins, the amino terminal methionine itself is subsequently removed by an aminopeptidase.
  • Binding of the 3OS ribosomal subunit at the ribosomal binding site is followed by assembly of a complete ribosome on the mRNA molecule. Once the ribosome has assembled at the Shine-Dalgarno sequence it scans the message until it encounters an initiator codon. Upon encountering the initiator codon the ribosome undergoes a conformational change and N-formylmethionine is inserted at the first position of the amino acid chain.
  • the ribosome After initiation the ribosome proceeds codon by codon along the mRNA inserting amino acids into the growing polypeptide chain. Termination is signaled by sequences encoded in the mRNA in the form of one of three stop codons, UAA, UAG or UGA. Upon reaching an in-frame stop codon, the ribosome falls off the mRNA and addition of amino acid residues to the polypeptide chain ceases.
  • the codons that serve as prokaryotic initiator codons are found not only at the initial position but also at other positions in the coding region.
  • each of these codons directs the insertion of N-formylmethionine at the initial position, none of them normally result in the insertion of N-formylmethionine when present at other positions.
  • the codons AUG, GUG, UUG, and AUU normally direct the insertion, respectively, of methionine, valine, leucine, and isoleucine.
  • tRNA Met Two classes of tRNA Met exist: tRNA p Met , which carries N-formylmethionine and tRNA ⁇ which carries methionine.
  • tRNA p Met which carries N-formylmethionine
  • tRNA ⁇ which carries methionine.
  • the ribosome takes part in a base pairing interaction between the 16S RNA component of the ribosome and the Shine-Dalgarno sequence. This interaction is thought to alter the conformation of the ribosome in such a way that the only transfer RNA that can bind to the 3OS subunit of the ribosome is tRNA F Met .
  • N-formylmethionine is then inserted as the initial amino acid of a polypeptide.
  • tRNA f Met When the Shine- Dalgarno induced interaction is not present, i.e., at an internal site, tRNA f Met does not bind to the ribosome.
  • the tRNA appropriate to the encountered codon (tRNA m M ⁇ t in the case of AUG, tRNA Val in the case of GUG, tRNA L ⁇ u in the case of UUG, and tRNA Il ⁇ in the case of AUU) binds to the ribosome with the concomitant insertion of its charged amino acid.
  • the invention depends on the observation that whether one of the four codons AUG, GUG, UUG, AND AUU functions as an internal codon depends on the presence or absence of an appropriately placed Shine-Dalgarno sequence.
  • the essential sequence requirements for the initiation of translation consists of an initiator codon downstream from a Shine-Dalgarno sequence.
  • Second site or internal initiation results in the production of a protein corresponding to the sequence between the second site start codon and the first in-frame stop codon encountered.
  • the invention features a method of preventing such undesirable initiation of translation at an internal initiator codon which is preceded by an internal Shine-Dalgarno sequence, by effecting a change in the DNA of either the internal initiator codon or the internal Shine-Dalgarno sequence.
  • the change preferably does not alter the amino acid sequence of the full length translation product.
  • the invention provides a number of methods for eliminating internal initiation.
  • the method employed in a particular application is dictated by the nature of the nucleotide sequence of the DNA encoding the desired full length desired translation product and the amino acid sequence of the desired full length translation product.
  • internal initiation is eliminated by effecting a change in the sequence of the internal initiator codon and in the Shine-Dalgarno sequence such that the internal initiator codon is converted to a codon that does not support initiation and such that any possibility of inappropriate ribosome binding is eliminated, all without changing the amino acid sequence of the full length translation product.
  • a change in the DNA sequence of the Shine-Dalgarno sequence is effected. This change is such that the functional Shine-Dalgarno sequence is destroyed without resulting in a change in the amino acid sequence of the full length translation product.
  • any change that will eliminate the existence of an internal initiator codon or destroy the superfluous functional Shine- Dalgarno sequence will result in a change in the sequence of the full length translation product.
  • a change in the DNA sequence is effected that will eliminate internal initiation and produce a polypeptide with the most conservative departure from the original full length translation product.
  • the invention improves yield of recombinant proteins and facilitates purification by preventing the production of non-functional truncated protein fragments beginning at internal initiation sites.
  • FIG. 1 is the sequence of the DNA expressing IL-2- toxin.
  • the internal GTG codon, starting at nucleotide 526, is indicated.
  • FIG. 2 is a codon dictionary.
  • FIG. 3 is a table of conservative amino acid substitutions.
  • FIG. 4 is a Shine-Dalgarno sequence in different registers with the in-frame codons of a mRNA molecule.
  • IL-2-toxin is a 68,170 dalton fusion protein expressed from a hybrid gene.
  • the recombinant gene contains both a portion of the diphtheria toxin gene and the interleukin- 2 (IL-2) gene.
  • DNA encoding the diphtheria toxin's generalized eukaryotic receptor binding domain is replaced with interleukin-2 (IL-2) encoding DNA, using recombinant DNA methods, as described in Murphy, U.S. Patent No. 4,675,382, hereby incorporated by reference.
  • IL-2-toxin can act as an IL- 2-receptor-positive-cell-destroying substance.
  • the purification of IL-2-toxin yields a 59,000 dalton polypeptide as a contaminant.
  • Amino acid sequence analyses of the copurifying polypeptide indicates that the N-terminal amino acid residue of the 59,000 dalton polypeptide is threonine, and thereafter the 59,000 dalton polypeptide is identical to the remaining 534 amino acid residues comprising the carboxyl terminal portion of IL-2-toxin.
  • the 59,000 dalton polypeptide is cross-reactive with anti-diphtheria toxin antibodies and anti-interleukin-2 antibodies. The antigenic determinants of these antibodies are both present in the region of IL-2-toxin corresponding to the sequence of the 59,000 dalton internal start polypeptide.
  • the sequence of IL-2-toxin is shown in FIG. l.
  • the 84th codon in the mRNA specifying IL-2-toxin is GUG, which encodes valine. (The 84th codon corresponds to nucleotides 526-528 in FIG. 1)
  • the 85th codon in the mRNA specifying IL-2-toxin is ACG, which encodes threonine.
  • the 59,000 dalton polypeptide contaminant is, we believe, derived from an internal initiation of translation at codon 84 in the IL-2-toxin mRNA.
  • GUG codon at position 84 Internal initiation of translation occurs at the GUG codon at position 84 probably because of the presence of a highly conforming representative of the Shine-Dalgarno sequence, UGGAGG, centered 13 bases 5 1 of the GUG codon. GUG may serve as an initiator codon in bacteria. Although GUG normally specifies valine, the Shine-Dalgarno sequence 5 1 to the GUG codon at position 84 results in initiation- ribosome binding, ribosomal assembly, and concomitant insertion of N-formylmethionine.
  • This N-formylmethionine residue which is the first amino acid of the internal start polypeptide, is cleaved by an aminopeptidase, and thus the initial amino acid residue of the ultimately recovered internal start contaminant is threonine.
  • This threonine corresponds to the threonine at position 85 of IL-2-toxin.
  • Second site initiation is undesirable for a number of reasons.
  • Second site starts may disrupt translation from properly initiated riboso es, as well as compete with initiation at the true initiation site for ribosomes, initiation factors, charged tRNAs, and any other factors needed for production of the final protein product.
  • second site initiators may reduce the overall yield of a desired product of protein synthesis.
  • the product of the second site starts may also result in the need for additional purification steps.
  • Products of internal initiation when in the same reading frame, can be very similar e.g., in size, physical properties and immunological specificity, to the desired product. These similarities can present particularly difficult purification problems.
  • Second site or internal initiation can be eliminated according to the invention by effecting an appropriate alteration in the sequence of either the codon being used as the second site initiator codon or in the nearest adjacent Shine-Dalgarno sequence or in both.
  • the most preferred alteration is one that, without altering the sequence of the desired translation product, is known unambiguously to be capable of eliminating internal initiation and ribosome binding. If only a single change can be made, changes at the internal initiator codon are thus preferable to changes at the internal Shine- Dalgarno, although changes at both sites is most preferable.
  • Changing the third "G” in the GUG codon at position 84 to "C” yields GUC, which encodes valine, but which cannot act as an initiator codon in the presence of a Shine- Dalgarno sequence.
  • the codon serving as the initiator codon of an internal start is UUG or AUU that is in-frame
  • UUG which normally encodes leucine
  • UUA which also encodes leucine but which cannot function as an initiator codon, even in the presence of a Shine- Dalgarno sequence
  • AUU which normally encodes isoleucine can be changed to AUC, which also encodes isoleucine but never serves as an initiator codon, even in the presence of a Shine-Dalgarno sequence.
  • second site initiation occurs at an in-frame AUG codon.
  • AUG is the only codon that directs the insertion of methionine.
  • a change at any of the three nucleotides of the AUG codon will result in the substitution of some other amino acid for methionine in the full length polypeptide.
  • Second site starts at in-frame AUG codons may be eliminated in one of three ways.
  • the functional integrity of the Shine- Dalgarno sequence and the internal initiator codon can be altered to eliminate internal starts.
  • the internal initiator alone can be altered.
  • the AUG serving as the second site initiator codon can be altered to a codon that does not support initiation.
  • any change in an AUG codon will be expressed in the full length polypeptide.
  • the choice of alterations should be limited to those resulting in an amino acid whose properties most resemble those of methionine.
  • FIG. 3 provides a table of the most conservative amino acid substitutions.
  • FIG. 4 depicts a Shine-Dalgarno sequence in each possible register with the in-frame codons of the corresponding polypeptides. Inspection of the dictionary of codons in FIG.
  • the internal initiator codon will be out-of-frame with the desired full length polypeptide.
  • an in-frame sequence specifying the insertion of isoleucine and cysteine could be comprised of AUA»UGU.
  • the final nucleotide of the isoleucine codon together with the first two nucleotides of the cysteine codon form an out-of-frame AUG.
  • alteration to yield the in-frame sequence AUC»UGU will replace the out-of-frame AUG with CUG, which never serves as an initiator codon.
  • the new sequence will result in the insertion of the same amino acids, isoleucine and cysteine, into the full length polypeptide.
  • it will not be possible to eliminate second site starts without effecting a change in the amino acid sequence of the full length polypeptide e.g., where the in-frame sequence AAA»UGU specifying lysine and cysteine creates an out-of-frame AUG formed from the last nucleotide of the lysine codon and the first two nucleotides of the cysteine codon.
  • the only change that can be made within the 6 nucleotides of the in-frame codons that is not expressed in the in-frame polypeptide is the change of the third nucleotide of the lysine codon (here A may be changed to G without a change in the polypeptide) .
  • This change destroys the out-of- frame AUG initiator codon but creates an out-of-frame GUG initiator codon.
  • second site starts may be eliminated by changes in the in-frame sequence that eliminate the out-of-frame initiator codon but result in only the most conservative changes in the amino acids inserted into the desired full length polypeptide.

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Abstract

Procédé de prévention d'un commencement indésiré de translation au niveau d'un codon initiateur interne dans une séquence d'ADN codant un polypeptide, consistant à placer une séquence Shine-Dalgarno en amont du codon initiateur interne. Le procédé consiste à modifier soit le codon initiateur interne soit la séquence Shine-Dalgarno, ou les deux, de sorte que l'on peut empêcher le commencement inapproprié de la translation et/ou de la liaison des ribosomes.
PCT/US1990/004113 1989-07-24 1990-07-20 Prevention d'un commencement de translation interne WO1991001374A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38424889A 1989-07-24 1989-07-24
US384,248 1989-07-24

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WO1991001374A1 true WO1991001374A1 (fr) 1991-02-07

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PCT/US1990/004113 WO1991001374A1 (fr) 1989-07-24 1990-07-20 Prevention d'un commencement de translation interne

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EP (1) EP0484411A4 (fr)
JP (1) JPH05501054A (fr)
AU (1) AU6067590A (fr)
CA (1) CA2063799A1 (fr)
NZ (1) NZ234616A (fr)
WO (1) WO1991001374A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070292918A1 (en) * 2006-05-30 2007-12-20 Stelman Steven J Codon optimization method
US8039230B2 (en) 2004-11-08 2011-10-18 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8263393B2 (en) 2002-12-20 2012-09-11 Chromagenics B.V. Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors
US20140186890A1 (en) * 2004-11-08 2014-07-03 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8999667B2 (en) 2004-11-08 2015-04-07 Chromagenics B.V. Selection of host cells expressing protein at high levels
US9228004B2 (en) 2004-11-08 2016-01-05 Chromagenics B.V. Selection of host cells expressing protein at high levels
US10317329B2 (en) 2015-10-09 2019-06-11 Genzyme Corporation Early post-transfection isolation of cells (EPIC) for biologics production
US11685943B2 (en) 2016-10-07 2023-06-27 Genzyme Corporation Early post-transfection isolation of cells (EPIC) for biologics production

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2007004595A (es) * 2004-11-08 2007-08-15 Chromagenics Bv Seleccion de celulas hospederas que expresan proteinas en altas concentraciones.
EP3134530B1 (fr) * 2014-04-25 2019-06-12 Genethon Traitement de la maladie de crigler et najjar

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biotechnology, Volume 3, issued August 1985, HALLING et al, "Expression in Escherichia coli of multiple products from a chimaeric gene fusion: evidence for the presence of procaryotic translational control regions within eucaryotic genes, pages 69-74, especially pages 72-73. *
Gene, Volume 72, issued January 1989, PREIBISCH et al, "Unexpected translation initiation within the coding region of eucaryotic genes expressed in Escherichia coli, pages 179-186, see page 180 and Table I. *
See also references of EP0484411A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8263393B2 (en) 2002-12-20 2012-09-11 Chromagenics B.V. Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors
US8039230B2 (en) 2004-11-08 2011-10-18 Chromagenics B.V. Selection of host cells expressing protein at high levels
US20140186890A1 (en) * 2004-11-08 2014-07-03 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8771984B2 (en) 2004-11-08 2014-07-08 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8999667B2 (en) 2004-11-08 2015-04-07 Chromagenics B.V. Selection of host cells expressing protein at high levels
US9228004B2 (en) 2004-11-08 2016-01-05 Chromagenics B.V. Selection of host cells expressing protein at high levels
US20070292918A1 (en) * 2006-05-30 2007-12-20 Stelman Steven J Codon optimization method
US10317329B2 (en) 2015-10-09 2019-06-11 Genzyme Corporation Early post-transfection isolation of cells (EPIC) for biologics production
US11635363B2 (en) 2015-10-09 2023-04-25 Genzyme Corporation FLARE (flow cytometry attenuated reporter expression) technology for rapid bulk sorting
US11685943B2 (en) 2016-10-07 2023-06-27 Genzyme Corporation Early post-transfection isolation of cells (EPIC) for biologics production

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Publication number Publication date
CA2063799A1 (fr) 1991-01-25
EP0484411A1 (fr) 1992-05-13
AU6067590A (en) 1991-02-22
NZ234616A (en) 1992-03-26
EP0484411A4 (en) 1992-06-17
JPH05501054A (ja) 1993-03-04

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