EP3052624A1 - Optimisation systématique de la séquence codant pour l'expression fonctionnelle d'une protéine - Google Patents

Optimisation systématique de la séquence codant pour l'expression fonctionnelle d'une protéine

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Publication number
EP3052624A1
EP3052624A1 EP13773223.6A EP13773223A EP3052624A1 EP 3052624 A1 EP3052624 A1 EP 3052624A1 EP 13773223 A EP13773223 A EP 13773223A EP 3052624 A1 EP3052624 A1 EP 3052624A1
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EP
European Patent Office
Prior art keywords
codon
expression
coding sequence
protein
host cell
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EP13773223.6A
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German (de)
English (en)
Inventor
John Van Der Oost
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Wageningen Universiteit
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Wageningen Universiteit
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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

Definitions

  • the present invention relates to a systematic approach to codon selection aimed at optimizing a coding sequence for functional expression of a heterologous protein in a host cell.
  • This approach recognizes that there is a certain codon landscape for optimal translation efficiency, and as such for maximal production of a protein.
  • This codon landscape consists of both optimal and non-optimal codons at certain positions in the nucleotide sequence, the pattern of which is hard to accurately predict with the current insights in the complex translation process.
  • the presented unbiased approach for optimization of the coding sequence relies on the provision of an expression library of synonymous coding sequences for the protein of concern. Expression of each such sequence in a given host cell is compared under certain conditions, desirably by high— throughput screening relying on screening for an optical signal.
  • Some features are general and can be easily manipulated by for example the choice of a suitable expression vector, e.g. adjusting promoter strength to enhance the transcription rate (DNA to mRNA), or by relatively straightforward genetic engineering , e.g. reducing any potential folding of the mRNA transcript around the translation start site in order to enhance the translation initiation process.
  • a suitable expression vector e.g. adjusting promoter strength to enhance the transcription rate (DNA to mRNA)
  • relatively straightforward genetic engineering e.g. reducing any potential folding of the mRNA transcript around the translation start site in order to enhance the translation initiation process.
  • some features are difficult to predict as they are specific for each gene or protein, for each host, as well as for the expression conditions.
  • One such variable is 'codon bias'.
  • Codon bias relates to the fact that some ('optimal') codons are well recognised by the corresponding tRNA and result in relatively fast translation; on the other hand, translation of other ('non-optimal') codons is much less efficient.
  • codon bias differs both within and between genomes and variation in codon optimisation amongst individual genes results in differential speed and accuracy in their translation (Rocha, 2004 Genome Res. 14: 2279-2286; Hershberg and Petrov, 2008 Annu. Rev. Genet 42: 287-299; Sharp et al., 2010 Philos. Trans. R Soc. Lond. B Biol. Sci. 365: 1203-1212 ). Research in the early 1980s revealed that several bacterial and yeast species are subject to translational selection, whereby highly-expressed genes
  • Organisms clearly differ both qualitatively and quantitatively with respect to their set of tRNAs, in the extent of codon usage bias across their genomes and in the forces determining it (Botzman and Margalit, 201 1 Genome Biol. 12: R109; Sharp et al., 2005 Nucleic Acids Res 33: 1141-1153).
  • heterologous expression may require adaptation of the codon bias to adjust it to match with the particular protein synthesis machinery of the production cell.
  • researchers have made attempts to understand the occurrence of 'non- optimal' codons.
  • the importance of 'non-optimal' (or 'slow') codons in loops for the successful co-translational folding of protein domains was demonstrated in Escherichia coii and Bacillus subtilis by Zhang et al. in 2009 ⁇ Nat. Struct. Mol. Biol. 16: 274-280).
  • the authors disclosed an algorithm to predict putative 'slow' translating regions in these species.
  • the method disclosed involves mapping the folding status of translation intermediates to determine whether local discontinuity in translation at certain regions in the mRNA sequence is needed to efficiently co-ordinate the rate of elongation of the peptide chain and its co-translational folding.
  • the algorithm is applied to the protein of interest to identify regions where 'non-optimal' codons may be required in order to allow slower translation and proper folding of the peptide.
  • the concentrations of iso- accepting tRNAs for a set of synonymous codons are variable, and the codon-reading programme can dramatically alter in response to both internal (gene expression level, GC content) and external factors (amino acid starvation, environmental stress, population size) (Elf et al., 2003 Science 300: 1718-1722; Subramaniam et al., 2013 PNAS 110: 2419- 2424; Behura and Severson, 2013 Biol. Rev. Camb. Philos. Soc 88: 49-61; Botzman and Margalit, 201 1 ). Variability in the codon reading programme may be particularly
  • the factors include: tRNA repertoire, codon position, GC content, expression level, gene length, amino acid conservation, transcriptional selection, RNA stability, protein hydrophobicity, recombination rates, environmental stress, population size, optimal growth temperature and organismal lifestyle have all been shown to be influential in determining the extent of codon bias (Chen et al., 2004 PNAS 101: 3480-3485; Hershberg and Petrov, 2009 PLoS Genetics 5: e1000556; Botzman and Margalit, 201 1 ; Behura and Severson, 2013; Akashi, 1997 Gene 205: 269-278; Powell and Moriyama, 1997 PNAS 94: 7784-7790; Moriyama and Powell, 1998 Nucleic Acids Res. 26: 3188-3193; Powell et al., 2003 J. Mol. Evol. 57 Suppl. 1: S214-225).
  • preferential codon usage is likely to be different between different genes, organisms, environmental situations and gene expression contexts.
  • this invention provides a systematic approach to generating high-levels of functional protein expression for any gene of interest, in a host cell of interest and under specific conditions of interest.
  • Adopting a non-prejudicial, function-oriented approach to generating high-levels of protein expression by systematically altering individual codons alone, or in combination, without affecting the polypeptide sequence enables attainment of a codon complement tailored to the production of optimal levels of functional protein in the cell of interest which takes account of the conditions for protein production, including the actual (rather than theoretical) tRNA status.
  • the present invention provides a method of codon selection for provision of a coding sequence for functional expression of a heterologous protein in a host cell, which, where the wild-type coding sequence is expressible in said host cell in an expression construct, comprises:
  • each variant coding sequence being inserted in an expression construct whereby each said sequence can be separately expressed in said host cell under the same conditions, and wherein each non-wild-type variant coding sequence differs from the wild-type coding sequence at one codon position, or at a cluster of sequential codon positions, or at a combination of codon sites selected from individual codon positions and clusters of sequential codon positions, the selected codon position(s) being a position or positions for which the host cell can provide more than one cognate tRNA and the variant coding sequences providing for each selected codon position both (a) the pre-determined optimal codon for translational efficiency and (b) one or more non-optimal synonymous codons;
  • the variant synonymous coding sequences provided will all differ from the wild- type coding sequence by just one codon and cover a number of selected codon positions. These may be all positions for which multiple cognate tRNAs are available in the host cell, although not necessarily so.
  • the expression library will include all possible synonymous codons.
  • step (i) of an expression library of synonymous coding sequences encoding said protein consisting of:
  • the variant coding sequences from the starting sequence will all exhibit just a single codon change compared to that sequence and cover a number of selected codon positions. All codon positions for which multiple cognate tRNAs are available may be included in the analysis, although again in some instances it may be chosen to cover less than the full complement of these. Again, for each selected codon position all possible synonymous codons may be provided.
  • a coding sequence derived by a method of the invention as above may be used as the starting sequence in substitution for said first coding sequence in a further cycle of codon selection.
  • This may, for example, comprise firstly provision of a set of variant sequences as defined above with the proviso that there is non-variance of one or more codons previously selected for substitution in the starting sequence.
  • each variant coding sequence of the expression library may incorporate codon
  • a cluster of sequential codon positions may be, for example, two adjacent codon positions or more than two adjacent codon positions, e.g. 3, 4, 5 or higher. Provision of an expression library for a further cycle of codon selection will be followed by repeat of steps (ii) and (iii) above. Further such cycles may be carried out.
  • the invention provides as its simplest mode for codon selection a method as follows resulting in provision of a coding sequence for functional expression of a heterologous protein in host cell. This method comprises:
  • each variant coding sequence being inserted in an expression construct whereby each said sequence can be separately expressed in said host cell under the same conditions, and wherein said variant coding sequences consist of:
  • these steps may be followed by one or more further rounds of codon selection as discussed.
  • the same method may be simply repeated but with one or more codons previously selected for substitution in the starting sequence being fixed. This may reveal optimal codon pairs and/ or clusters.
  • determination of optimal functional protein expression may take account of need for co-expression of one or more further
  • heterologous proteins in the same host cell at a desired level. Where only expression of a single heterologous protein is of concern, then codon selection will simply be on the basis of association with the highest functional expression of the desired protein.
  • a coding sequence may be provided for the protein of interest wherein each position for which synonymous codons can be used is either the pre-determined optimal codon or in preference a synonymous non-optimal codon which has been found to correspond with improved functional protein expression.
  • the provision of the coding sequence may comprise actual synthesis of a sequence comprising the coding sequence. This may be for example by de novo synthesis or possibly by modification of a pre-existing sequence, e.g. by site-directed mutagenesis. Subsequent expression of the coding sequence in the chosen host cell may be carried out.
  • the sequence will preferably be provided in an expression construct, e.g.
  • Figure 1 shows a hypothetical example of the systematic manipulation of individual codons and high-throughput screening of a library of synonymous variants to generate a sequence for improved functional protein expression in a specific production host.
  • a suitable model system for this approach is expression of green fluorescent protein (GFP) as a model protein in E. coli as a model host since functional expression of the model protein can be simply directly detected by fluorescence measurement.
  • GFP green fluorescent protein
  • Other models include proteins with established 3D structure that allow for easy functional screening and for which functional production has been established in the chosen host cell.
  • the essential starting point for a method of codon optimization according to the invention is a coding sequence which expresses the protein of interest and can be expressed to some degree as a heterologous protein in the chosen host cell.
  • the wild type sequence may be utilized as the starting sequence.
  • an expression library may be provided of synonymous coding sequences including the wild-type sequence and wherein each variant non-wild-type coding sequence differs from the wild-type coding sequence at one codon position, or at a cluster of sequential codon positions, or at a combination of codon sites selected from individual codon positions and clusters of sequential codon positions.
  • the variant coding sequences will provide for each selected codon position (a position for which there is more than one cognate tRNA) both (a) the pre-determined optimal codon for translation efficiency and (b) one or more non-optimal synonymous codons. All possible codons will generally be provided for each selected position.
  • a different coding sequence may be chosen as the starting point for codon optimization.
  • This may conveniently be a synthetic sequence of pre-determined optimal codons, e.g. a commercially available coding DNA of pre-determined optimal codons or such a sequence with one or more non-optimal substitutions based on prior
  • an alternative starting expression library for codon optimization may be one in which the variant coding sequences consist of:
  • codons will commonly be provided for each selected position.
  • more than one round of codon optimization may be carried out using a method of codon selection according to the invention. All codon positions for which more than one cognate tRNA is available may be included in a round of analysis.
  • a common starting point will be an expression library in which compared to the starting sequence (either the wild-type sequence or first coding sequence as defined above) each variant sequence has just a single codon substitution and preferably the full length of the coding sequence is covered. It will be appreciated that where there is a starting methionine (Met) codon this will remain ATG in all variants since there are no synonymous codons (see Fig.1 ). However in some instances a different start codon may be present in the starting sequence or substituted.
  • Met methionine
  • prior information may mean that more directed codon substitutions may be made covering less than the full length sequence.
  • native protein coding sequences in that species commonly have a 'starting ramp' of about 10 codons long which directs low translation efficiency and is thought to be beneficial for overall functional expression of the corresponding proteins (Pechman and Frydman, ibid).
  • it may be chosen to apply the approach of the invention to codon optimization at a section of a protein coding sequence downstream of a 5' starting section of a plurality of codons, e.g. at least 10 codons, at least 20, 30, 40 or 50 codons, which may be kept in all variants tested as the wild-type sequence.
  • Each variant coding sequence may be conveniently generated by site-directed
  • pre-determined optimal codon may be equated with any of the following:
  • the approach of the invention can be applied to any type of host cell which can be cultured, and which is genetically accessible (i.e. able to serve as a host for functional production of the protein of interest (POI)). It may be applied to all host cells commonly employed for recombinant heterologous protein expression including bacterial cells (e.g. E. coli, Bacillus subtilis), yeast cells (e.g. S. cerevisiae, Pichia pastoris), fungal cells, insect cells and mammalian cell lines (e.g. CHO cells) and tumour cell lines (e.g HeLa cells).
  • the POI may be a native protein of a host cell in which the native coding sequence for that protein has been knocked out. In these circumstances, the POI will be considered as a heterologous protein to the mutated host cell.
  • the expression constructs of the library may be located in plasmids (expression vectors) which are used to transform the host cell.
  • Methods of transformation may include, but are not limited to, heat shock, electroporation, particle bombardment, chemical induction, microinjection and viral transformation.
  • the expression levels of the protein for each synonymous coding variant are determined under the same expression conditions.
  • functional expression e.g. as with GFP or by enzymatic action of the protein of interest (POI) to generate a detectable optical signal.
  • POI protein of interest
  • the POI will be detectable by a high-throughput screening method, for example, relying on detection of an optical signal.
  • a tag may be, for example, a fluorescence reporter molecule translationally-fused to the C-terminal end of the POI, e.g. GFP, Yellow Fluorescent Protein (YFP), Red Fluorescent Protein (RFP) or Cyan Fluorescent Protein (CFP). It may be an enzyme which can be used to generate an optical signal.
  • Tags used for detection of expression may also be antigen peptide tags.
  • a tag may be provided for affinity purification, e.g. a His tag.
  • the codon selected from amongst synonymous codons for any selected position will be the codon associated with the highest or optimal observed functional expression of the POI, or where more than one codon provides substantially equal such expression, one such codon corresponding with that level of expression. Where there is choice of codons indicated for a selected position based on the expression data, preference may be given to the codon in the starting sequence, i.e. the wild type codon if the starting sequence is the wild-type sequence. This will minimise the number of codon changes to convert the starting sequence in a nucleic acid to the selected synonymous coding sequence for improved functional protein expression.
  • POIs may preferably be recovered from the cell culture medium as secreted proteins, although they may also be recovered from host cell lysates.
  • a method of the invention for codon optimization may be repeated with one or more further proteins, all the selected proteins for heterologous expression sharing one or more structural motifs.
  • the analysis carried out may further include determination of whether any signature pattern of optimal and non-optimal codons is associated with any shared structural motif.
  • the invention also extends to synthesizing a protein coding sequence including a signature pattern so determined and expressing the sequence, preferably in the same host cell and under the same expression conditions as employed for codon optimization.
  • a method of providing a DNA comprising a coding sequence for expressing a heterologous protein in a host cell which includes incorporating in said sequence a pattern of optimal and non-optimal codons at a site associated with provision of a structural motif, wherein said pattern enables increased expression efficiency of said protein in said host cell compared with the synonymous coding sequence containing solely optimal codons, wherein optimal codons are those codons pre-calculated to provide the highest codon translation efficiency in the host cell or the sole possible codon.
  • the DNA may be provided for example by site-directed mutagenesis or de novo synthesis and once obtained, the coding sequence for the protein of interest may desirably be expressed in said host cell.
  • GFP provides a suitable model protein for exemplification of codon optimization according to the invention starting with a wild-type coding sequence, since the protein is well characterized and its functional expression can be simply assayed by high throughput fluorescent screening in E. col i as a model host. Screening can be done either
  • Figure 1 illustrates generation of a synthetic library of synonymous coding sequences wherein the variants each have an initiating Met codon and each variant of the wild-type sequence has one codon change. For each codon position for which synonymous codons are available all possible codons are provided in the library.
  • each variant under the same conditions The fluorescence associated with functional expression of each variant under the same conditions is determined. For each codon position for which multiple codons are present in the library, a codon is selected which gives the highest expression. Where one such codon is the wild-type codon, this is maintained in the final selected sequence.
  • the wild type sequence is supplemented by 3 variant synonymous coding sequences to provide all 4 codons for the amino acid Gly.
  • the variant providing the non-wild type codon GGC at codon position 2 is observed to give the highest fluorescence and is selected for codon position 2 in the new coding sequence.
  • codon position 3 just one variant coding sequence is supplied to supplement the wild- type coding sequence so as to provide in the expression library both possible codons for Asp at that position.
  • the variant gives no significant difference in the level of fluorescence and thus the wild-type codon is maintained in preference in the new coding sequence.
  • the same approach to codon selection is repeated for other codon positions.
  • the selected complete new sequence may be obtained for expression by site-directed mutagenesis of the wild-type sequence.
  • the new selected sequence may be used as the starting sequence for one or more further rounds of codon optimization in accordance with the invention aimed at revealing optimal codon pairs and/ or clusters.
  • the above method of the example may be repeated but with one initially selected codon at a variant codon position fixed.

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Abstract

Cette invention concerne une approche systématique concernant le choix des codons pour préparer la séquence codant pour l'expression fonctionnelle d'une protéine hétérologue chez une cellule hôte. Cette approche reconnaît que, pour l'efficacité de la traduction, des codons non optimisés peuvent être souhaitables en certains sites et repose sur l'utilisation d'une banque d'expression de séquences codantes synonymes pour la protéine concernée. L'expression de chacune de ces séquences est comparée dans les mêmes conditions chez la cellule hôte, de préférence par un criblage à haut débit.
EP13773223.6A 2013-10-02 2013-10-02 Optimisation systématique de la séquence codant pour l'expression fonctionnelle d'une protéine Withdrawn EP3052624A1 (fr)

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WO2023026292A1 (fr) * 2021-08-25 2023-03-02 Ramot At Tel-Aviv University Ltd. Expression optimisée dans des organismes cibles

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US10724040B2 (en) 2015-07-15 2020-07-28 The Penn State Research Foundation mRNA sequences to control co-translational folding of proteins
GB201600512D0 (en) * 2016-01-12 2016-02-24 Univ York Recombinant protein production
EP3363900A1 (fr) * 2017-02-21 2018-08-22 ETH Zurich Assemblage d'adn multiplexe guidé par l'évolution de pièces d'adn, voies et géomes
CN113195719A (zh) * 2018-09-26 2021-07-30 卡斯西部储备大学 用于增加蛋白质表达和/或治疗单倍剂量不足症的方法及组合物
FR3099179A1 (fr) * 2019-07-22 2021-01-29 Universite De Rennes 1 Methode pour determiner l’effet d’une mutation sur l’expression d’un gene d’interet

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WO2023026292A1 (fr) * 2021-08-25 2023-03-02 Ramot At Tel-Aviv University Ltd. Expression optimisée dans des organismes cibles

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