JP2008521421A - Lupac bifunctional marker and its use in protein production - Google Patents

Abstract

  The present invention relates to the industrial production of proteins. More particularly, the present invention relates to a Lupac surrogate marker corresponding to a fusion protein of luciferase and puromycin N-acetyltransferase. The present invention further relates to a method of screening for cells that highly express a protein of interest using Lupac.

Description

  The present invention relates to the industrial production of proteins. More particularly, the present invention relates to alternative markers corresponding to the fusion of luciferase and puromycin N-acetyltransferase. The present invention further relates to a method for screening cells that highly express a protein of interest using this alternative marker.

  Introducing a heterologous gene into an animal host cell and screening for expression of the added gene is a long and complex process. There are typically a number of hurdles to overcome. The hurdles are (i) large expression vector composition: (ii) stable transfection over time, and finally no selection pressure, transfection and selection of clones; (iii) high expression of heterologous proteins of interest Screening.

  1． Selection of clones expressing heterologous genes

  Selection of clones with an integrated gene of interest is performed using a selectable marker that confers resistance to selection pressure. Many selectable markers confer resistance to antibiotics (eg, neomycin, kanamycin, hygromycin, gentamicin, chloramphenicol, puromycin, zeocin, or bleomycin).

  When generating a cell clone that expresses a gene of interest from an expression vector, a host cell is typically transfected with a plasmid DNA vector that encodes both the protein of interest and a selectable marker on the same vector. The acceptability of the plasmid is often very limited, and the selectable marker must be expressed from a second plasmid that is co-transfected with that plasmid containing the gene of interest.

  Stable transfection causes the expression vector to be randomly integrated into the host cell genome. Utilizing selective pressure (eg, administering antibiotics to the media) will remove all cells that have not incorporated the respective antibiotic or a vector containing a selectable marker that confers resistance to selective pressure. If this selectable marker is on the same vector as the gene of interest, or if this selectable marker is on a second vector and simultaneously incorporates a vector containing the gene of interest, the cell Will express both genes. However, it has often been observed that the level of expression of the gene of interest varies greatly depending on where the site of integration is.

  In addition, when selective pressure is removed, expression often becomes very unstable or even disappears. Therefore, it is very cumbersome to identify such clones in a large candidate population, since only a few initial transfectants provide high levels and stable long-term expression. Typically, high expression candidates are isolated and then cultured in the absence of selective pressure. Under such conditions, the first selected candidate large population is removed, since the expression of the gene of interest is impaired when the selection pressure is removed. Therefore, it may be advantageous to cultivate candidates in the absence of selective pressure after the initial selection period for stable transfection and then only screen for the gene of interest.

  2． Screening for high expression clones

  Screening for clones that highly express the protein of interest is often done by a method that directly indicates that the protein is present in large quantities. Typically, immunological methods (such as ELISA or immunohistochemical staining) are applied to detect the product intracellularly or in the supernatant of the cell culture. These methods are tedious, expensive, time consuming, and often do not allow high throughput screening (HTS). In addition, antibodies that react to the expressed protein must be available.

  Attempts have been made to quantify proteins by fluorescence activated cell sorting (FACS), but only in limited cases (especially secreted proteins) (Borth et al., 2000).

  One approach to screening for proteins of interest with high expression rates would be to use an easily measurable alternative marker that is expressed from the same vector as the gene of interest (Chesnut et al., 1996). The idea behind the use of measurable surrogate markers is that the gene of interest and surrogate marker genes on the same vector are physically linked, so the expression of these two genes is correlated It is.

  Numerous markers are readily available in the art that can be easily measured. These markers usually act on chromogenic or luminescent substrates such as β-glucuronidase, chloramphenicol acetyltransferase, nopaline synthase, β-galactosidase, secreted alkaline phosphatase (SEAP), and luciferase. Corresponds to the enzyme. Green fluorescent protein (GFP) can also be used as a measurable marker in FACS. The activity of all these proteins can be measured by standard assays that can be used with HTS.

  The disadvantage of this approach is the use of yet another expression cassette for the alternative marker gene. Thus, the expression vector containing the expression unit containing the promoter, cDNA, and polyadenylation signal for at least three proteins (ie, gene of interest, selectable marker, and surrogate marker) is quite large. For multi-chain proteins, the situation is further complicated. Alternatively, separate plasmid vectors expressing three genes each encoding a protein of interest, a selectable marker, and an alternative marker can be co-transfected. However, each vector may be integrated into a different locus, or the degree of expression may vary and not be correlated.

  One promising approach to solving the above constraints is to use a chimeric marker that combines the function of a selectable marker with the function of a measurable marker. Such an approach has been published, for example, by Bennett et al. (1998). This paper discloses a GFP-Zeo ™ marker that confers resistance to the antibiotic zeocin. The expression of this marker can be monitored with a fluorescence microscope.

  EP1 262 553 discloses a chimeric marker and a method for using the chimeric marker in a method for capturing an unknown gene or selecting a cell in which a genetic element has been integrated into a given locus by homologous recombination. ing. However, EP 1 262 553 does not teach the use of chimeric markers to screen for clones that express high levels of recombinant protein. Furthermore, the experimental data relates to a chimeric marker corresponding to a fusion protein between luciferase and a protein that confers resistance to hygromycin.

  The feasibility of using chimeric markers for screening high-expressing clones remains largely unexplored, and the effectiveness of screening high-expressing clones using such chimeric markers needs further study. There is. The discovery of new, alternative, and strong chimeric alternative markers would be very useful in the field of industrial production of therapeutic proteins.

  The present invention is based on the construction and characterization of Lupac, a novel bifunctional chimeric marker. Lupac corresponds to a fusion protein between luciferase and puromycin N-acetyltransferase (pac), a protein that confers resistance to puromycin. Lupac has been demonstrated to combine the functional attributes of both luciferase and pac. Furthermore, Lupac has also been demonstrated to be useful in isolating high expressing clones for therapeutic proteins.

  Accordingly, a first aspect of the present invention includes a luciferase fragment fused to a puromycin N-acetyltransferase (pac) fragment, comprising (i) luciferase activity; and (ii) puromycin N-acetyltransferase activity. The Lupac polypeptide as indicated.

  A second aspect relates to a nucleic acid encoding a Lupac polypeptide according to the present invention.

  A third aspect relates to a vector comprising the nucleic acid according to the present invention.

  A fourth aspect relates to a cell comprising the nucleic acid according to the present invention.

  A fifth aspect relates to the use of a cell comprising a nucleic acid according to the invention for producing a protein of interest.

  The sixth aspect relates to the use of the polypeptides, nucleic acids, and vectors according to the present invention for screening for cells that express the protein of interest.

The seventh aspect relates to a method for screening a cell that expresses a protein of interest, the method comprising the steps of: (i) transfecting a cell with an expression vector according to the present invention;
(Ii) selecting a cell resistant to puromycin; and (iii) examining the luciferase activity of the cell selected in step (ii).

The eighth aspect relates to a method of obtaining a cell line that expresses a protein of interest,
(I) screening the cells according to the screening method of the present invention;
(Ii) selecting a cell that exhibits the highest expression of the protein of interest; and (iii) establishing a cell line from the cell.

The ninth aspect relates to a method for producing a protein of interest, the method comprising:
(I) culturing the cell line obtained according to the present invention under conditions that allow the protein of interest to be expressed; and (ii) recovering the protein of interest.

A tenth aspect relates to a method for producing the polypeptide of the present invention, and the method comprises:
(I) culturing cells containing the nucleic acid of the invention under conditions that allow the Lupac polypeptide to be expressed; and (ii) recovering the polypeptide according to the invention.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO: 1 corresponds to the nucleic acid sequence of the Lupac polypeptide according to the invention.
SEQ ID NO: 2 corresponds to the protein sequence encoding the Lupac polypeptide according to the invention.
SEQ ID NOs: 3-6 correspond to the primers used to construct the Lupac polypeptide according to the present invention.
SEQ ID NO: 7 corresponds to a fragment of the mouse CMV immediate early region present in the pBSI.IL18BPmCMVLupac.I vector.
SEQ ID NO: 8 corresponds to the protein sequence of photinus pyralis luciferase.
SEQ ID NO: 9 corresponds to the protein sequence of pac of Streptomyces alboniger.

  The present invention is based on the construction and characterization of a novel bifunctional chimeric marker called Lupac. Lupac corresponds to a fusion protein between luciferase and puromycin N-acetyltransferase (pac), a protein that confers resistance to puromycin.

  Lupac has been demonstrated to combine the features of both luciferase and pac functions (Example 2). Thus, the Lupac marker can be used both as a selectable marker in stable transfection due to its pac activity and as an alternative marker that can be easily measured due to its luciferase activity.

  Furthermore, it was demonstrated that Lupac is useful for isolating clones that highly express therapeutic proteins. In Example 3, a vector containing Lupac expressed from two different promoters and the protein of interest was constructed. It was shown that there was a very good positive correlation between the expression level of Lupac and the expression level of the gene of interest.

  Thus, the present invention provides Lupac, a powerful marker. This Lupac can be used to provide selectivity in stable transfection, while also functioning as an alternative marker for screening candidate clones that highly express the gene of interest. When Lupac is used in HTS, the possibility of selecting a highly expressing clone can be maintained to the same extent as when directly measuring the expression level of a gene of interest. In addition, using Lupac can save time, reduce costs, and reduce materials. This is because (i) standardized independently of the product and simple analysis is made; and (ii) measurement of luciferase activity is an inexpensive assay.

  1． Lupac polypeptide

  A first aspect of the present invention includes a luciferase fragment fused to a puromycin N-acetyltransferase (pac) fragment, and exhibits (i) luciferase activity; (ii) puromycin N-acetyltransferase activity Relates to Lupac polypeptides. Hereinafter, the term “Lupac polypeptide” or “Lupac” will be referred to as such a polypeptide.

As used herein, a polypeptide exhibits “luciferase activity” when it can oxidize luciferin. It is preferable that the polypeptide can serve as a catalyst for at least one of the following reactions.
Firefly (Photinus) luciferin + O ₂ + ATP => oxidized firefly luciferin + CO ₂ + H ₂ O + AMP + diphosphate + light.
Renilla or Cypridina luciferin + O ₂ <=> Oxidized luciferin + CO ₂ + light.

  Firefly luciferin refers to (S) -4,5-dihydro-2- (6-hydroxy-2-benzothiazoloyl) -4-thiazolecarboxylic acid. Cypridina luciferin is [3- [3,7-dihydro-6- (1H-indol-3-yl) -2-[(S) -1-methyl-6-propyl] -3-oxoimidazo- [1 , 2-a] pyrazin-8-yl] propyl] guanidine, Renilla luciferin is 8-benzyl-2- (4-hydroxybenzyl) -6- (4-hydroxyphenyl) imidazo- [1,2 -A] means pyrazin-3 (7H) -one.

  By measuring the light generated during the above reaction, the activity of luciferase can be measured. Luciferase activity can be measured, for example, as published in Example 2.2.

  As used herein, a polypeptide exhibits “puromycin N-acetyltransferase activity” when it can confer resistance to puromycin to a cell. Puromycin N-acetyltransferase activity can be measured, for example, as published in Example 2.3.

  In preferred embodiments, the Lupac polypeptide comprises a fragment of luciferase from a firefly (eg, photinuspyralis, Luciola cruciata, Luciola lateralis, or Photuris pennsylvanica) . Preferably, the Lupac polypeptide contains a fragment of a firefly luciferase. As used herein, the term “North American firefly luciferase” refers to the polypeptide of SEQ ID NO: 8, or an allelic variant thereof, or a splice variant thereof, or a mutein thereof. Most preferably, the fragment of the firefly luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8. Alternatively, the Kita firefly luciferase fragments are at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 of the full-length firefly luciferase. , 325, 350, 375, 400, 425, 450, 475, 500, or 525 amino acid fragments, or the full length luciferase of North American firefly.

  In other preferred embodiments, the Lupac polypeptide comprises a fragment of luciferase from Renilla reniformis (sea pansy) or Vargula hilgendorfii (sea firefly). Yes.

  In other preferred embodiments, the Lupac polypeptide comprises a fragment of pac from the genus Streptomyces (eg, Streptomyces alboniger, or Streptomyces coelicolor). The Lupac polypeptide preferably contains a pac fragment of Streptomyces alboniger. As used herein, the term “Streptomyces alboniger pac” means the polypeptide of SEQ ID NO: 9, or an allelic variant thereof, or a splice variant thereof, or a mutein thereof. More preferably, this pac fragment contains amino acids 2-199 of SEQ ID NO: 9. Alternatively, the Streptomyces alboniger pac fragment is a fragment of at least 50, 75, 100, 125, 150, or 175 amino acids of the full-length pac of Streptomyces alboniger, or Streptomyces alboniger It may correspond to the full length pac.

  In a Lupac polypeptide, the luciferase fragment can be fused to the 5 ′ end of the pac fragment, or the pac fragment can be fused to the 5 ′ end of the luciferase fragment. Preferably, the luciferase fragment is fused to the 5 ′ end of the pac fragment.

  In the most preferred embodiment, the Lupac polypeptide comprises or consists of SEQ ID NO: 2.

  In other optimal embodiments, the Lupac polypeptide has an amino acid sequence that is at least 50% identical to SEQ ID NO: 2, more preferably an amino acid sequence that is at least 60% identical, even more preferably at least 70%, 75%, 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99% contain identical amino acid sequences or consist of such amino acid sequences.

  As used herein, the term “mutein” refers to a natural polypeptide in which one or more amino acid residues of a natural polypeptide are replaced with another amino acid residue. One or more amino acid residues are deleted or one or more amino acid residues are added to the natural polypeptide, but the activity of the resulting product is greatly reduced compared to the natural polypeptide It means something that is not. Such muteins are made by known synthetic methods and / or site-directed mutagenesis techniques, or by any other suitable known technique. A mutein that can be used in the present invention for Streptomyces alboniger pac or Kita firefly luciferase, or a nucleic acid encoding this mutein, including substantially corresponding sequences such as substituted peptides and substituted polynucleotides Would be routinely obtained by one of ordinary skill in the art without undue experimentation based on the suggestions and instructions presented in this specification.

  The muteins of the present invention relating to Streptomyces alboniger pac or Kita firefly luciferase include conditions of moderate or high stringency under the DNA or RNA of the present invention encoding pac or luciferase. Proteins encoded by nucleic acids that hybridize with (eg, DNA or RNA) are included. The term “stringent conditions” refers to conditions commonly referred to as “stringent” by those of ordinary skill in the art, among hybridization and subsequent washing conditions. Ausubel et al., Current Protocols in Molecular Biology, Interscience, NY, §6.3 and 6.4 (1987, 1992) and Sambrook et al. (Sambrook, JC, Fritsch, EF, Maniatis, T., 1989, Molecular Cloning: A laboratory Manual, Cole Spring Harbor Laboratory Press, Cold Spring Habor, NY).

  Non-limiting examples of stringent conditions include 2 × SSC and 0.1% SDS for 5 minutes in 2 × SSC and 0.5% SDS at a temperature 12-20 ° C. below the calculated Tm of the hybrid under test. Wash for 15-60 minutes at 37 ° C. in 0.1 × SSC and 0.5% SDS for 30-60 minutes and then at 68 ° C. in 0.1 × SSC and 0.5% SDS for 30-60 minutes Includes conditions. One of ordinary skill in the art will understand that stringent conditions also depend on the length of the DNA sequences, oligonucleotide probes (eg, 10-40 bases), mixed oligonucleotide probes. When using a mixed probe, it is preferable to use tetramethylammonium chloride (TMAC) instead of SSC.

  The Streptomyces alboniger pac or Kita firefly luciferase mutein is at least 50% identical to the natural polypeptide, more preferably at least 60% identical, and even more preferably at least 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98%, or 99% polypeptides having the same amino acid sequence.

  For example, a polypeptide having an amino acid sequence that is at least 95% “identical” to the reference amino acid sequence of the present invention includes an amino acid sequence of the subject polypeptide that contains up to 5 different amino acids for every 100 amino acids in the reference amino acid sequence It means that it is identical to the reference sequence except that it may be present. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% (5 out of 100) amino acid residues of the target sequence are inserted or May be deleted, or substituted with other amino acids.

  “% Identity” can be determined for multiple sequences for which no exact correspondence exists. In general, the alignment is such that the correlation between the two sequences to be compared is maximized. This may include operations to improve the degree of alignment by inserting “gaps” into one or both sequences. The percent identity can be determined over the entire length of each sequence being compared (so-called overall alignment). This is particularly suitable for sequences of identical or similar length. Alternatively, the% match can be determined for a shorter predetermined length (so-called local alignment). This is more suitable for sequences of different lengths.

  Methods for comparing the identity and homology of two or more sequences are well known in the art. For example, determine the% identity of two polynucleotides using programs available in the Wisconsin Sequence Analysis Package, Version 9.1 (Devereux et al., 1984) (eg BESTFIT and GAP), or% identity of two polypeptide sequences Gender and% homology can be determined. BESTFIT (Smith and Waterman, 1981) uses a “local homology” algorithm that finds the single closest region between two sequences. Other programs for determining identity and / or similarity between sequences are also known in the art, such as the BLAST family of programs (Altschul et al., 1990, accessible through the NCBI home page on the World Wide Web site). Year) and FASTA (Pearson and Lipman, 1988; Pearson, 1990).

  Preferred changes for muteins of the present invention are what are known as “conservative” substitutions. Conservative amino acid substitutions in Streptomyces alboniger pac or Kita firefly luciferase have synonymous amino acids within one group. Synonymous amino acids have physicochemical properties that are close enough to each other so that the biological function of the molecule is preserved when members of the group are interchanged (Grantham, 1974). Obviously, amino acid insertions and deletions to the above sequence are possible without changing the function of the sequence. In particular, amino acids (eg cysteine residues) in which insertions or deletions are only a few amino acids (eg less than 30 and preferably less than 10) and are very important for a functional conformation This is true if the is not removed or moved. Proteins and muteins produced by such deletions and / or insertions are within the scope of the present invention.

  The group of synonymous amino acids is preferably that defined in Table I. More preferably, the group of synonymous amino acids is as defined in Table II. Most preferably, the group of synonymous amino acids is that defined in Table III.

  Examples of protein amino acid substitution methods that can be used to obtain muteins for use in the present invention with respect to Streptomyces alboniger pac or Kita firefly luciferase are any known methods, such as the United States granted to Mark et al. Patents 4,959,314, 4,588,585, 4,737,462; US Pat. No. 5,116,943 to Koths et al .; US Pat. No. 4,965,195 to Namen et al .; US Pat. No. 4,879,111 to Chong et al .; Lee In US Pat. No. 5,017,691; lysine substituted proteins are presented in US Pat. No. 4,904,584 (Shaw et al.).

  Preferably, the muteins of the present invention exhibit substantially the same biological activity as the corresponding natural polypeptide.

  2． Lupac nucleic acids and vectors and host cells containing them

  A second aspect of the present invention relates to a nucleic acid encoding a Lupac polypeptide. In the present specification, such nucleic acids are hereinafter referred to as “Lupac nucleic acids”.

  In a preferred embodiment, the Lupac nucleic acid comprises or consists of SEQ ID NO: 1.

  Any method known in the art can be used to obtain the Lupac nucleic acid of the present invention. Lupac nucleic acid is obtained, for example, as described in Example 1.

  The third aspect of the present invention relates to a vector containing a Lupac nucleic acid. Such vectors are referred to herein as “Lupac vectors”. The Lupac vector is preferably an expression vector. Such vectors are further referred to as “Lupac expression vectors”. The term “Lupac vector” also includes “Lupac expression vector”. The term “vector” is used herein to refer to a double- or single-stranded circular or linear DNA or RNA compound, which includes a host cell, ie, a single cell or a multicellular. At least one polynucleotide of the present invention to be transferred to a host organism. An “expression vector” includes the appropriate signal within the vector. This signal includes a variety of regulatory elements such as enhancers / promoters that express polynucleotides derived from viruses, bacteria, plants, mammals, and other eukaryotes and inserted into host cells.

  In an optimal embodiment, the Lupac expression vector further comprises a nucleic acid encoding the protein of interest. As shown in Example 3, such vectors are particularly useful for screening cells that highly express the protein of interest.

  In the present invention, a “protein of interest” may be any polypeptide whose production is desired. The protein of interest will find use in the field of medicine, agricultural business, or laboratory instruments. The protein of interest is preferably used in the pharmaceutical field.

  The protein of interest may be, for example, a naturally secreted protein, a normal cytoplasmic protein, a normal transmembrane protein, or a human or humanized antibody. If the protein of interest is a normal cytoplasmic protein or a normal transmembrane protein, it is preferably genetically engineered to make the protein soluble. The polypeptide of interest may be of any origin. The polypeptide of interest is preferably of human origin.

  In a preferred embodiment, the protein of interest is selected from chorionic gonadotropin, follicle stimulating hormone, lutropin-chorionic gonadotropin, thyroid stimulating hormone, human growth hormone, interferon (eg, interferon β-1a, interferon β-1b ), Interferon receptors (eg interferon gamma receptors), TNF receptors p55 and p75, interleukins (eg interleukin-2, interleukin-11), interleukin binding proteins (eg interleukin-18 binding proteins), anti CD11a antibody, erythropoietin, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, pituitary peptide hormone, climacteric gonadotropin, insulin-like growth factor (eg somatomedin-C), keratinocyte growth factor, glial cell line derived neurotroph Child, thrombomodulin, basic fibroblast growth factor, insulin, factor VIII, somatropin, osteoinductive factor-2, platelet-derived growth factor, hirudin, epoetin, recombinant LFA-3 / IgG1 fusion protein, glucocerebrosidase, and These muteins, fragments, soluble forms, functional derivatives, and fusion proteins are made of the group.

  In a preferred embodiment, the Lupac expression vector is a nucleic acid encoding the protein of interest and includes at least two promoters. One promoter expresses the Lupac polypeptide and the other promoter expresses the protein of interest. Such vectors may further comprise enhancer regions and / or expression enhancing sequences (eg, insulators, border elements, LCRs (eg, Blackwood and Kadonaga, described in 1998)) or matrix / scaffold binding regions (eg, described in Li et al., 1999). There may be an internal ribosome entry site (IRES) between the different ORFs in the Lupac expression vector.

  Alternatively, the Lupac expression vector contains a promoter that drives both the expression of the protein of interest and the expression of Lupac. The Lupac ORF is separated from the ORF of the protein of interest by the presence of IRES. IRES may be derived, for example, from viral or cellular genes.

  As used herein, the term “promoter” refers to a region of DNA that functions to control transcription of one or more DNA sequences, and to bind and interact with a DNA-dependent RNA polymerase binding site. Structurally identified by the presence of other DNA sequences that regulate promoter function. The functional expression promoting fragment of a promoter is a shortened promoter sequence or a truncated promoter sequence that retains the activity as a promoter. Promoter activity is determined by any assay known in the art (eg, a reporter assay using luciferase as the reporter gene (Wood et al., 1984; Seliger and McElroy, 1960; de Wet et al., 1985)); or Promega ( Commercially available from the trademark)). An “enhancer region” is a region having a function of increasing transcription of one or more genes in a DNA region. More specifically, the term “enhancer” as used herein is a DNA regulatory element that promotes, augments, enhances, or improves the expression of a gene regardless of position or orientation with respect to that gene to be expressed; The expression of one or more promoters can be promoted, increased, enhanced, or improved.

  In a preferred embodiment, the Lupac expression vector comprises at least one promoter of the mouse CMV immediate early region. This promoter may be, for example, the mCMV IE1 gene promoter described in WO 87/03905 and known (“IE1 promoter”). The promoter may be the promoter of the mCMV IE2 gene (“IE2 promoter”). The mCMV IE2 gene itself is known from, for example, a paper by Messerle et al. (1991). The IE2 promoter region and IE2 enhancer region are described in detail in PCT / EP2004 / 050280. The Lupac expression vector preferably contains at least two promoters of the murine CMV immediate early region. More preferably, the two promoters are an IE1 promoter and an IE2 promoter. Optimally, the Lupac expression vector comprises SEQ ID NO: 7 containing therein an IE1 promoter, an IE2 promoter, and an enhancer region.

  In a preferred embodiment, the Lupac expression vector contains at least two promoters of the murine CMV immediate early region. One promoter expresses the Lupac polypeptide and the other promoter expresses the protein of interest. This embodiment is realized by the pBSI.IL18BPmCMVLupac.I vector shown in FIG. 4, wherein the IE1 promoter contained therein expresses the Lupac polypeptide, and the IE2 promoter expresses the protein of interest.

  In another preferred embodiment, the promoter of the murine CMV immediate early region expresses the gene encoding the protein of interest, and the Lupac polypeptide is expressed from another expression cassette inserted into the vector backbone. The IE1 and IE2 promoters express either one of the same two copies of the gene encoding the protein of interest, or two subunits of the multimeric protein of interest (eg, antibody or peptide hormone) be able to.

  In another preferred embodiment, the Lupac expression vector comprises an amplification marker. The amplification markers include, for example, adenosine deaminase (ADA), dihydrofolate reductase (DHFR), multidrug resistance gene (MDR), ornithine decarboxylase (ODC), and N- (phosphonacetyl) -L-aspartate resistance (CAD) ). Amplifying the gene encoding the protein of interest can increase the level of expression of that protein of interest when the vector is incorporated into a cell (Kaufman et al., 1985).

  A fourth aspect of the present invention relates to cells transfected with a Lupac vector. Many cells are suitable for the present invention, such as primary cell lines and established cell lines from a wide variety of eukaryotes (including plant cells and animal cells). Suitably the cell is a eukaryotic cell. More preferably, the cell is a mammalian cell. Optimally, the cells are Chinese hamster cells or human cells.

  Suitable cells include, for example, NIH-3T3 cells, COS cells, MRC-5 cells, BHK cells, VERO cells, CHO cells, rCHO-tPA cells, rCHO-Hep B surface antigen cells, HEK 293 cells, rHEK cells, rC127- Hep B surface antigen cells, CV1 cells, mouse L cells, HT1080 cells, LM cells, YI cells, NS0 and SP2 / 0 mouse hybridoma cells, RPMI-8226 cells, WI-38 cells, normal human fibroblasts, humans There are stromal cells, human hepatocytes, human osteosarcoma cells, Namalwa cells, human retinoblasts, PER.C6 cells, and other immortalized and / or transformed cells of mammals.

  3． How to use Lupac

  The fifth aspect relates to a method for producing a protein of interest using cells containing Lupac nucleic acid. Preferably said cell comprises a Lupac vector.

  As discussed in Examples 3.3.2, 3.3.3, and 4, the use of Lupac polypeptide as a selectable / alternative marker offers many advantages in screening cells that highly express the protein of interest. . In particular, since the expression of Lupac polypeptide is highly correlated with the expression of the protein of interest, it is advantageous to first screen for Lupac that is highly expressed. The expression of the protein of interest is examined in the second screening. This screen is performed only with the best producer cells isolated in the first screen looking for highly expressed Lupac.

  Therefore, the sixth aspect of the present invention relates to a method for screening a cell expressing a protein of interest or a cell highly expressing using a Lupac polypeptide, a Lupac nucleic acid, or a Lupac expression vector. The first screening on the cells looks for Lupac that is highly expressed, then inference correlates Lupac expression with the expression of the protein of interest. This quickly removes 80-95% of the cells tested because of the low Lupac expression level, allowing the remaining 5-20% to be analyzed in the second step for the expression of the gene of interest. .

  With respect to the methods and methods of use of the present invention, “high expression” means that the level of expression in the particular cell screened is higher than the level of expression in the other cells screened. The “high expression” of a protein is a relative value. For example, the final expression level of a commercially produced recombinant protein ranges from 1 to 2,000 mg / l, depending on the protein, the annual requirement and the therapeutic dose. During screening, the expression level of the protein of interest is lower than the final expression level.

The seventh aspect relates to a method for screening a cell that expresses a protein of interest or a cell that highly expresses the method,
(I) transfecting a cell with a Lupac expression vector;
(Ii) selecting a cell resistant to puromycin; and (iii) examining the luciferase activity of the cell selected in step (ii).

  In a preferred embodiment, 20% of the cells that exhibit the highest luciferase activity in step (iii) contain the cells with the highest expression of the protein of interest. Preferably, 10% of the cells exhibiting the highest luciferase activity in step (iii) contain the cells with the highest expression of the protein of interest. Optimally, 1 or 5% of the cells exhibiting the highest luciferase activity in step (iii) contain the cells with the highest expression of the protein of interest.

  Luciferase activity is preferably measured by the Bright-Glo Luciferase assay using a Centro LB 960 photometer during the acquisition time of 5 seconds in step (iii) of the above method.

  Any number of cells can be screened by such methods. Preferably, the luciferase activity of at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 cells is examined in step (iii). Optimally, screening a population of cells sufficient to obtain at least 1,000 to 10,000,000 independent transfectants resistant to puromycin. Of these cells, luciferase activity can be further examined for at least 10 to 1,000,000 candidate clones resistant to puromycin.

  Cells obtained at the end of the above screening method can be ranked relative to the expression of Lupac. Cells that exhibit the highest luciferase activity when any of the above methods are complete can be selected. For example, individual cells that exhibit luciferase activity corresponding to the top 5-20% of Lupac-expressing cells are selected and the expression of the gene of interest is further analyzed in subsequent steps.

  In a preferred embodiment, the above screening method further comprises a step (iv) of selecting from about 1% to about 20% of the cells examined in step (iii). The selected cell is the cell that exhibits the highest luciferase activity in step (iii). Based on the highest luciferase activity, about 5% to about 20% of the cells examined in step (iii) can be selected. Alternatively, on the basis of the highest luciferase activity, about 1%, 1.5%, 2%, 3%, 4%, 5% to about 30%, 40%, 50%, 60 examined in step (iii) %, 70% and 80% of cells can be selected.

  In another preferred embodiment, the above screening method is performed using a multi-well microtiter plate, or the like.

  When a cell exhibiting the highest luciferase activity is selected, the expression level of the protein of interest contained in the selected cell can be further examined.

Therefore, the eighth aspect relates to a method of obtaining a cell line that expresses a protein of interest,
(I) screening the cells according to any of the above screening methods;
(Ii) selecting a cell with the highest expression of the protein of interest; and (iii) establishing a cell line from the cell.

  As used herein, “cell line” means one particular type of cell that can be grown in a laboratory. Cell lines can usually be grown in permanently established cell cultures and will grow indefinitely if given fresh media and space appropriately. Methods for establishing cell lines from isolated cells are well known to those skilled in the art.

A ninth aspect relates to a method for producing a protein of interest, which comprises (i) culturing the cell line obtained as described above under conditions that allow the protein of interest to be expressed; and (ii) And) recovering the protein of interest.

  Conditions that allow the expression of the protein of interest can be readily established by those skilled in the art by standard methods. For example, the conditions disclosed in Example 3.3.1 can be used.

  In a preferred embodiment, the above method for producing a protein of interest further comprises the step of purifying said protein of interest. Purification may be performed by any method known to those skilled in the art. When the protein of interest is used in the pharmaceutical field, the protein of interest is preferably formulated into a pharmaceutical composition.

The tenth aspect relates to a method for producing a Lupac polypeptide, the method comprising: (i) culturing a cell containing a Lupac nucleic acid under conditions capable of expressing the Lupac polypeptide; and (ii) recovering the Lupac polypeptide. Comprising the steps of:

  Such a method may be performed, for example, as published in Example 2. Such methods can further comprise purifying the Lupac polypeptide by any known method in the art.

  Having fully described the invention herein, the invention can be practiced with a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation. Will be understood by those skilled in the art.

  Although the present invention has been described in a particular manner, it will be understood that further modifications are possible. This application generally covers any variations, uses, or modifications in accordance with the principles of the invention and is well-known or commonly used in the technical field to which the invention pertains, and in the specification of the appended claims. It is intended to include deviations from the present disclosure that may be applied to the essential features described.

  All references cited herein (eg, journal articles and abstracts, US published patent applications, unpublished patent applications, foreign patent applications, patents granted in the United States or foreign countries, and any other references) , Including all data, tables, drawings, and texts presented in the cited references, are hereby incorporated by reference in their entirety. Further, the references cited in the references are also incorporated in this specification as a whole.

  When referring to individual steps of a known method, individual steps of a conventional method, known methods, conventional methods, any aspect, description, or embodiment of the invention is disclosed, taught, or suggested in the related art. It never admits that it will be done.

  The above description of specific embodiments will fully reveal the overall features of the invention, so that others will be familiar with the knowledge of those skilled in the art, including the contents of the references cited in this specification. Can be easily modified and / or modified for various applications without undue experimentation and without departing from the general concept of the present invention. Would be able to. Accordingly, such alterations and modifications are intended to be within the meaning and scope equivalent to the disclosed aspects, based on the teachings and guidance presented herein. The terminology or expressions in this specification are for the purpose of illustration and are not intended to limit the invention, and such terms or expressions in this specification usually follow the teachings and instructions provided herein. In combination with the knowledge of those skilled in the art.

Lupac configuration
1.1. Lupac Nucleic Acid Acquisition by PCR Lupac was constructed by fusing firefly luciferase and PAC open reading frame by PCR cloning.

  The template DNA for luciferase was the pGL3-ctrl plasmid (Pronega, catalog # E1741), and the template DNA for puromycin acetyltransferase was the pPUR plasmid (Clontech, catalog # 6156-1). Two pairs of PCR primers (SEQ ID NOs: 3-6) were designed to allow amplification and fusion of the 3 ′ end of luciferase to the 5 ′ end of puromycin acetyltransferase. The first PCR primer amplifies from the PpuMI site of the luciferase gene to the last amino acid, excluding the stop codon, and the second PCR primer, except for the first methionine, which functions as the translation start codon. The 'end was fused to the 5' end of puromycin acetyltransferase.

PCR conditions were as follows:
Amplification of luciferase: 50 pmoles of primers with SEQ ID NOs: 3 and 4; 20 ng of pGL3-ctrl; 200 μM of each dNTP, 1 × Thermopol buffer, Vent DNA polymerase (New England Biolabs, catalog # M0254S, 10 × buffer) 2 units) with 5% DMSO for a total volume of 50 μl.

  -Amplification of pac: Same chemical conditions as luciferase except that the primers of SEQ ID NOs: 5 and 6 were 50 pmol and pPur was 20 ng.

·cycling:
-Denaturation: 1 cycle, 98 ° C, 5 minutes
-Hybridization / polymerization: 25 cycles of 1 minute at 98 ° C and 1 minute at 60 ° C
-Final polymerization: 1 cycle, 72 ° C, 10 minutes.

  The PCR for amplifying luciferase yielded a 474 base pair band, while the PCR for amplifying pac gave a 618 base pair band.

  Each PCR reaction was purified using a Macherey-Nagel Nucleospin extraction kit (Catalog # 740 590.50) according to the manufacturer's protocol.

Another PCR reaction was performed as follows using 1 μl of each purified PCR fragment.
The primers with SEQ ID NOs: 3 and 6 were used at 50 picomoles. The same mixture as described for PCR in the first step was used except that Vent DNA polymerase was added after the first denaturation step (hot start method). The cycling parameters remained essentially the same except that the hybridization / polymerization temperature was raised to 65 ° C.

  Two DNA fragments can be fused into a single 1092 base pair fragment by PCR reaction.

  The PCR fragment was purified by cutting the band from the agarose gel and centrifuged in a Corning filter chip (Catalog # 4823) for 10 minutes at 9600 rpm in an Eppendorf table centrifuge. The eluate was then precipitated by adding 2 volumes of 100% ethanol and centrifuged at full speed.

1.2. Cloning of the nucleic acid
1.2.1. pBS-Lupak
The pellet was resuspended and treated with T4 polynucleotide kinase (Stratagene, catalog # 600103) according to the manufacturer's protocol. The recipient vector was pBluescript II SK (+) (Catalog # 212205-01) cut with EcoRV and then treated with calf intestinal alkaline phosphatase (Gibco 18009-027) according to the manufacturer's protocol. Plasmid pBS-Lupac was obtained by conventional ligation and cloning in E. coli. The sequence of this plasmid was then confirmed by sequencing.

1.2.2. pGLupac
The confirmed pBS-Lupac sequence was digested with PpuMI / XbaI, and a 1.1 kb band was purified using a Corning chip and ethanol precipitation. The recipient vector was pGL3-ctrl digested with PpuMI / XbaI. A 4.8 kb band was purified by elution with a Corning chip. The vector pGLupac obtained after ligation contained the Lupac sequence of SEQ ID NO: 1. This Lupac sequence encodes the Lupac polypeptide of SEQ ID NO: 2.

  pGLupac is shown in FIG. pGLupac comprises an ORF for Lupac expressed from the SV40 promoter with an SV40 enhancer at the 3 ′ end.

1.2.3. pGLupac-Basic
pGL3 basic (Promega, catalog # E1751) was cut with NcoI / XbaI. pGLupac was cut with NcoI / XbaI and the 2.3 kb Lupac insert was purified using a Corning chip. The vector obtained after ligation was named pGLupac-basic.

1.2.4. pBSI.IL18BPmCMVLupac.I
pGLupac-basic was digested with NheI / ClaI / PvuI. A 2.6 kb NheI / ClaI fragment was purified. The recipient vector was obtained by digesting a vector containing the mouse CMV immediate early region with NheI and ClaI. This recipient vector contains the DNA sequence of SEQ ID NO: 7 containing the IE1 and IE2 promoters, as well as the IE1 and IE2 enhancers. A detailed description of the function of the DNA sequence of SEQ ID NO: 7 is described in PCT / EP2004 / 052591. The recipient vector further contained a DNA sequence encoding IL18BP (SwissProt accession number O95998).

  The vector obtained after ligation was named pBSI.IL18BPmCMVLupac.I. pBSI.IL18BPmCMVLupac.I is shown in FIG. IL18BP expression is driven by the IE2 promoter, whereas Lupac expression is driven by the IE1 promoter.

  In this construct, IL18BP corresponds to the protein of interest to be produced and Lupac corresponds to an alternative marker.

Characterizing the function of Lupac An assay was performed to determine whether Lupac provides two functions (ie, measurable luciferase activity and resistance to puromycin) in stably transfected cells. This was investigated by transfecting CHO cells with an expression vector for Lupac. As a control, CHO cells co-transfected with a firefly wild-type luciferase expression vector and a pac expression vector were used.

2.1. Transfection of CHO cells with lipofectamine
CHO-S cells were purchased from Gibco / Invitrogen (catalog number: 11619).
Format: 6-well plate CHO-S cells in exponential growth phase were diluted 24 hours prior to transfection to give 0.75 × 10 ⁶ cells per ml.

0.6 × 10 ⁶ cells with 4.5 mM glutamine and 1 × hypoxanthine / thymidine (HT) (100 × HT, Invitrogen, catalog number: 11067-030; L-glutamine, 200 mM, Sigma, G-7513) Resuspended in supplemented 440 μl ProCho5 medium (Cambrex, catalog number: 12766Q).

Two types of mixtures were prepared.
Mixture A: Lipofectamine (Invitrogen, catalog number: 18324-012): 8.8 μl
ProCho5 medium: 211.2 μl
Total volume: 220μl
Mix B: 1 μg of plasmid DNA each for control or Lupac constructs.
Control: 0.5 μg pGL3-ctrl + 0.5 μg pPur.
Lupac: 0.5 μg pGLupac + 0.5 μg irrelevant plasmid (pBluescript II SK (+)).
ProCho5 medium: supplemented to 220 μl.

Mixture A and Mixture B were mixed and left at room temperature for 30 minutes. This A + B mixture was added to 440 μl medium containing 0.6 × 10 ⁶ cells. The resulting cell suspension was placed in a 37 ° C., 5% CO ₂ incubator for 3 hours, and 1.6 ml of ProCho5 supplemented with 1 × HT and 4.5 mM L-glutamine was added. This cell suspension was further cultured under the same conditions.

2.2. Luciferase measurement:
2.2.1. Protocol Luciferase activity was measured 2 days after transfection. The Bright-Glo Luciferase assay system was purchased from Promega (Cat. No. E2610). The assay was performed according to the manufacturer's guidelines. In brief, the cell suspension was homogenized by pipetting up and down several times and 50 μl aliquots were taken and placed in white 96-well plates (Nunc, catalog number: 236108). 50 μl of reconstituted Bright-Gro reagent was added directly and the cell suspension was incubated at room temperature for 5 minutes. Luminescence was measured using a Centro LB 960 photometer (Berthold Technologies) with an acquisition time of 5 seconds. Luminescence is measured in units of relative light units (RLU). Results were normalized with respect to cell number (cell density in units of 1 million cells / ml).

2.2.2. Results The results are shown in FIG. Luciferase activity was measured in both cells transfected with pGLupac and cells transfected with pGL3-ctrl + pPur. As a result, Lupac exhibits luciferase activity.

2.3. Selection of resistance to puromycin
2.3.1. Protocol After measuring luciferase activity, the remaining cells from the 6-well plate were transferred to a 15 ml Falcon tube, centrifuged, and the cell pellet was transferred to 5% fetal calf serum (FBS) in another 6-well plate. ) Was resuspended in 2 ml of medium. Forty-eight hours after transfection, selection was performed by replacing the medium with ProCho5 / HT / glutamine / 5% FBS containing 10 μg / ml puromycin (Sigma, P-8833). Every 2 days old media was discarded, washed with 1 × PBS, and fresh selective media was added. Two weeks after selection, cells are trypsinized, counted, serially diluted, and 1000, 500, 100, 50, 20 cells per well in a 6-well format , So that it becomes 10 pieces. Ten days later, colonies that grew in all dilutions were counted, and all were picked and grown in serum-free suspension to analyze protoclones.

  In a positive control co-transfected with pGL3-ctrl and pPur, 57 clones grew.

  In the negative control corresponding to untransfected CHO-S wild type cells, no clones grew.

  When pGLupac and pBluescript II SK (+) were transfected, 48 clones grew.

  Therefore, it was observed that a similar amount of clones grew in the selective medium. As a result, it can be concluded that the puromycin resistance provided by the fusion protein is comparable to the puromycin resistance provided by the wild-type puromycin resistance gene.

  Next, the luciferase activity of the resistant clones was measured. By luciferase measurement, the percentage of clones that express both functions when transfected with pGLupac is greater than the clone that expresses both functions when luciferase and puromycin resistance genes are transfected simultaneously on separate vectors. Many were found (Table IV). This confirms the effect of Lupac.

  In conclusion, the Lupac fusion protein exhibits the combined activity and function of luciferase and pac protein.

Use of Lupac as an alternative marker The two functions of the generated Lupac fusion protein suggest that it has two effects. First, Lupac should allow isolation of stably transfected clones by their puromycin resistance. Second, Lupac expression should reflect the expression level of the gene of interest physically linked by measurement of luciferase activity. To test this hypothesis, a series of clones was generated from a pool of cells stably transfected with pBSI.IL18BPmCMVLupac.I. Lupac activity and IL18BP expression levels were measured.

3.1. Transfection of CHO cells with lipofectamine
CHO-S cells were purchased from Gibco / Invitrogen (catalog number: 11619).
Format: T75 flask.
CHO-S cells in exponential growth phase were subcultured up to 24 hours before transfection. The cells were diluted to 0.75 × 10 ⁶ cells per ml.

Among the T75 flasks, 5 × 10 ⁶ cells, 1 × HT (Invitrogen, Catalog No. 11067-030) and 4.5mM L- glutamine (Sigma, Cat #: G-7513) in 7ml supplemented with Resuspended in ProCho5 medium (Cambrex, catalog number: 12766Q).

Two types of mixtures were prepared.
Mixture A: Lipofectamine (Invitrogen, catalog number: 18324-012): 52.1 μl
ProCho5 medium: 517.9 μl
Total volume: 570μl
Mixture B: DNA: 5 μg of pBSI.IL18BPmCMVLupac.I linearized with XmnI and 5 μg of irrelevant plasmid (pBluescript II SK (+)) for a total of 10 μg of DNA
ProCho5 medium: supplemented to 570 μl.

Mixture A and mixture B were combined and incubated at room temperature for 30 minutes.
This A + B mixture was added to 7 ml of medium containing 5 × 10 ⁶ cells. This suspension was returned to the incubator at 37 ° C. and 5% CO ₂ and left for 3 hours. The culture was then centrifuged at 800 g for 3 minutes and the cell pellet was resuspended in 5 ml ProCho5 supplemented with 1 × HT and 4.5 mM L-glutamine. Next, 5 ml of ProCho5 / HT / glutamine was added to the suspension by adding it directly to the T75 flask. Finally, 5 × 10 ⁶ cells were present in 10 ml of ProCho5 / HT / glutamine medium.

3.2. Selection procedure 48 hours after transfection, the medium is changed and cells are brought to 0.5 × 10 ⁶ cells / ml in ProCho5 / HT / glutamine containing 10 μg / ml puromycin (Sigma, P-8833). The selection was made by diluting to Cells were counted every 2 days, centrifuged, and resuspended in fresh selective medium so that there were 0.5 × 10 ⁶ cells / ml in living cells. Survival was checked every 2 days. After 21-35 days, the selection was finished. The survival rate again reached over 80% after the expected initial decline.

  Once a stable pool has been established, use a multi-drop dispenser (ThermoLabsystem, Cat. No. 5840150) to place the cells in a 384-well plate (Nunc, Cat. No.) at a density of one cell per well (70 μl / well). 164688) and two weeks later, randomly picked clones were rearranged in one 96-well plate.

3.3. Expression analysis
3.3.1. Protocol A high-throughput format was used (96-well plates) to assess expression of both genes.

  Day 1: Cells were diluted to 50% in 100 μl fresh ProCho5 containing 100 μl ProCho5 culture medium (serum free) + 5% fetal calf serum. Maintenance plates were subcultured weekly after dilution to 1/20 in serum-free ProCho5 medium.

  -Day 2: The medium was discarded and washed once with 200 μl of 1 × PBS (Invitrogen, catalog number: 10010-015). 75 μl of fresh ProCho5 containing 5% FBS was added and the resulting suspension was incubated for a 24 hour expression pulse period.

  Day 3: 50 μl of supernatant was collected and 200 μl of ELISA buffer (1 × PBS, 0.1% w / v BSA, 0.2% v / v Tween 20) was added. 100 μl was analyzed by a standard ELISA assay for IL18BP.

  The wells were washed with 200 μl 1 × PBS (discard) and 100 μl Glo lysis buffer (Promega, E266a) was added. The wells were incubated for 30 minutes at room temperature to ensure cell lysis.

  Luciferase measurements were performed using 30 μl of lysed cells transferred to white 96-well plates (Nunc, catalog number: 236108) +30 μl of reconstituted Bright-Gro reagent (Promega, E263a). Luminescence was measured using a Centro LB 960 photometer with an acquisition time of 5 seconds.

  In FIG. 4, the expression level of IL18BP is measured in response units (RU, an indicator provided by the manufacturer) by the Biacore instrument, and luciferase activity is measured in relative light units (RLU).

3.3.2. result
85 candidate clones were randomly selected and examined for the expression of both Lupac and IL18BP. 24 clones were selected for further investigation. Each of the 8 clones represents a clone that highly expresses Lupac, a clone that expresses moderately, and a clone that expresses lightly. Next, the expression of both genes in all 24 of these clones was examined again.

  FIG. 4 shows the correlations observed in these experiments. There is a very good correlation between Lupac expression and IL18BP expression, especially at both ends of the range. In other words, the clone that most highly expresses Lupac corresponds to the clone that most highly expresses IL18BP.

  Therefore, using Lupac as a selectable / alternative marker, candidate clones can be established and screened using vectors that express both Lupac and the protein of interest. For example, when the first screening for searching for highly expressed Lupac is performed, clones that highly express the gene of interest can also be selected with high probability. Thus, screening using ELISA or other methods that are very tedious, time consuming and costly can be avoided. Furthermore, since Lupac screening is independent of the particular gene of interest selected, the same approach can be used for a variety of screening programs. This is an obvious logical advantage. The expression of the gene of interest will only be examined when performing a second screen on the largest production clone from the first screen.

3.3.3. Conclusion
When Lupac is used in HTS, the probability of selecting a high expression clone can be kept the same, and time and materials can be reduced. In classic HTS clone production approaches, the best clones are typically selected based on high titer secreted proteins when screening over 2,000 clones. Using Lupac, clones that gave similar productivity for IL18BP were obtained when only 85 clones were screened. The reduction in the number of samples screened to obtain a gene of interest with similar productivity makes it easier to use the Lupac approach, which is accompanied by sampling errors and assay variations in ELISA high-throughput screening. It may be related to the decrease. Furthermore, by selecting 5-10 best clones per plate, it is expected that the best one clone will be selected per plate. Thus, using Lupac to screen 1,000 clones would reduce the number of clones to be analyzed to 50-100 and consequently avoid the second HTS.

  Furthermore, it is important to note that, as explained herein, the fusion of two independent enzymes with very different activities and origins surprisingly retains their function in Lupac. . Clearly, having two functions will have two effects. This is because Lupac can be used to provide selectivity in stable transfection and to serve as an alternative marker for screening candidate clones that highly express the gene of interest.

  Finally, it is worth noting in this experiment that intracellular Lupac expression is highly correlated with secreted IL18BP expression. Other high-throughput techniques (eg, cell sorting using FACS) have obvious limitations in screening secreted proteins.

Acquisition of clones producing a large amount of r-SP1 The purpose of this experiment was to develop a CHO cell line that produces a large amount of recombinant protein called recombinant Serono protein 1 (r-SP1).

  There was no direct high-throughput screening method for r-SP1 protein. A commercially available ELISA assay for measuring the amount of r-SP1 in a sample could only analyze 8 samples per microtiter plate due to its low sensitivity. Such an ELISA assay is referred to as a “low-throughput” ELISA assay as opposed to a “high-throughput” ELISA assay that can analyze nearly 96 samples per microtiter plate.

  Screening clones that produce high r-SP1 using a commercially available low-throughput ELISA assay would be a very tedious and time-consuming method (if that is practical). The following shows that instead of using such a method that takes a long time, a high-producing clone could be screened quickly and successfully using Lupac as an alternative marker.

4.1. Experimental Approach for Screening A flow chart of the experimental approach is shown in Table 5.
FIG. 5 shows an expression vector encoding the recombinant protein r-SP1 and combined with other elements necessary for effective expression of the bidirectional mouse CMV IE1 promoter and mouse CMV IE2 promoter. This vector expresses r-SP1 and Lupac simultaneously. A pool of stably transfected cells was established in the presence of puromycin.

  After selection, r-SP1 titers and specific production rates (pcd) were assessed at the pool level, and clone isolation was performed on the best two pools.

  700 clones for each pool selected were analyzed twice in a high-throughput luciferase assay. A clone expressing the highest level of Lupac was selected. This allowed us to reduce the number of clones from 1400 to 19 in just two days.

  These 19 clones were further analyzed by quantitative ELISA to determine r-SP1 titer and specific productivity, and clones that expressed the highest level of r-SP1 were selected. These clones were further cloned once to ensure that independent clones were obtained. The specific productivity (pcd) of the independent clone was analyzed. A master cell bank was established for the most promising clones. The resulting cell line was named CHO-r-SP1.

Table 5
Construct expression vector ↓
Transfect expression vectors into CHO cells ・ Suspension culture in serum-free medium ↓
Establish a stable pool ・ Select in the presence of puromycin ↓
Isolate 1400 candidate clones • Ultradilute to one cell per well in a 384 well plate • Relocate candidate clones from a 384 well plate to a 96 well plate ↓
Screen in 96-well plates using a high-throughput luciferase ELISA assay • 1400 candidate clones → 19 candidate clones in just 2 days ↓
Measure r-SP1 expression using low-throughput r-SP1 ELISA assay ・ 19 candidate clones → 5 candidate clones ↓
Reclon 5 candidate clones ↓
Evaluate clones ↓
Select the clone that produces the best ↓
Configure the master cell bank ↓
Develop method and produce r-SP1

  4.2. Production of r-SP1 from CHO-r-SP1 cell line

  The CHO-r-SP1 cell line was cultured using a fed-batch method in suspension in serum-free medium in a 250 liter bioreactor. As shown in FIGS. 6 and 7, the titer of r-SP1 reached a high level of 401 mg / l after 22 days. Cell survival at the end of the experiment was still good. The CHO-r-SP1 cell line grew to a high cell density (10 million cells / ml) and the specific productivity average was 2 pcd.

  4.3. Conclusion

  This experiment confirms the use of Lupac in an actual screening format. Using Lupac as a surrogate marker, production clones expressing high levels of the protein of interest could be successfully selected, which was not possible with direct high-throughput screening methods.

References

The alignment of Lupac polypeptide (sequence ID number 2), luciferase (sequence ID number 8), and pac (sequence ID number 9) of this invention is shown. A diagram of the pGLupac vector and the pBSI.IL18BPmCMVLupac.I vector is shown. pGLupac contains an ORF for the Lupac polypeptide of SEQ ID NO: 1 expressed from the SV40 promoter with an SV40 enhancer at the 3 ′ end. Plasmid pBSI.IL18BPmCMVLupac.I contains the Lupac polypeptide of SEQ ID NO: 2 expressed from the IE1 promoter of the two-way mouse CMV immediate early region. The IE2 promoter of this vector expresses IL18BP. The luciferase activity of CHO cells transiently transfected with pGLupac or pGL3-ctrl + pPur (vectors each containing luciferase and pac) is shown. Luciferase activity is standardized at cell density (100,000 cells / ml). FIG. 5 shows that there is a positive correlation between the expression (RLU) of the Lupac polypeptide of SEQ ID NO: 2 and the expression (RU) of IL18BP in 24 clones transfected with the pBSI.IL18BPmCMVLupac.I vector. FIG. 5 is a diagram of an expression vector used in Example 4. This vector contains the Lupac polypeptide of SEQ ID NO: 2 expressed from the IE1 promoter in the immediate early region of the bi-directional mouse CMV. The IE2 promoter of this vector expresses a recombinant protein called “Serono protein 1” (r-SP1). Insulators are described in PCT / EP2004 / 052591. The r-SP1 titer and the viable cell density obtained when culturing a CHO cell line expressing r-SP1 (CHO-r-SP1) are shown. Such a CHO cell line was selected using Lupac as an alternative marker (see Example 4). The r-SP1 titer and the viable cell density obtained when culturing a CHO cell line expressing r-SP1 (CHO-r-SP1) are shown. Such a CHO cell line was selected using Lupac as an alternative marker (see Example 4).

Claims (39)

A fragment of luciferase fused to a fragment of puromycin N-acetyltransferase (pac),
(I) luciferase activity;
(Ii) a Lupac polypeptide exhibiting puromycin N-acetyltransferase activity.   2. The Lupac polypeptide of claim 1, wherein the luciferase is a photinus pyralis luciferase.   The Lupac polypeptide of claim 2, wherein the fragment of luciferase comprises amino acids 1 to 547 of SEQ ID NO: 8.   4. The Lupac polypeptide according to any one of claims 1 to 3, wherein the pac is a Streptomyces alboniger pac.   The Lupac polypeptide according to claim 4, wherein the fragment of pac comprises amino acids 2-199 of SEQ ID NO: 9.   6. The Lupac polypeptide according to any one of claims 1 to 5, wherein the fragment of luciferase is fused to the 5 'end of the fragment of pac.   6. The Lupac polypeptide according to any one of claims 1 to 5, wherein the fragment of pac is fused to the 5 'end of the fragment of luciferase.   7. The Lupac polypeptide of claim 6, comprising SEQ ID NO: 2.   A nucleic acid encoding the Lupac polypeptide according to any one of claims 1-8.   9. The nucleic acid of claim 8, comprising SEQ ID NO: 1.   A vector comprising the nucleic acid according to claim 9 or 10.   12. The vector according to claim 11, which is an expression vector.   13. The expression vector according to claim 12, further comprising a nucleic acid encoding a protein of interest.   14. An expression vector according to claim 13, comprising at least two promoters, one expressing the Lupac polypeptide and the other expressing the protein of interest.   15. The expression vector according to claim 14, wherein the at least two promoters are murine CMV immediate early region promoters.   16. The expression vector according to claim 15, wherein the at least two promoters are an IE1 promoter and an IE2 promoter.   Selected from the group consisting of adenosine deaminase (ADA), dihydrofolate reductase (DHFR), multidrug resistance gene (MDR), ornithine decarboxylase (ODC), and N- (phosphonacetyl) -L-aspartate resistance (CAD) The expression vector according to any one of claims 12 to 16, further comprising an amplification marker.   A cell comprising the nucleic acid according to claim 9 or 10.   The cell according to claim 18, comprising the vector according to any one of claims 11 to 17.   20. The cell according to claim 18 or 19, which is a mammalian cell.   21. The cell of claim 20, which is a CHO cell.   21. The cell of claim 20, which is a human cell.   23. Use of a cell according to any one of claims 18 to 22 for producing a protein of interest.   Use of a Lupac polypeptide according to any one of claims 1 to 8 for screening cells expressing a protein of interest.   Use of the nucleic acid according to claim 9 or 10 for screening cells expressing the protein of interest.   Use of the vector according to any one of claims 12 to 17 for screening cells expressing a protein of interest.   27. Use according to any one of claims 24 to 26, wherein the expression of the protein of interest is correlated with the activity of luciferase. A method for screening cells expressing a protein of interest,
(I) transfecting a cell with the expression vector according to any one of claims 12 to 17;
(Ii) selecting a cell resistant to puromycin; and (iii) examining the luciferase activity of the cell selected in step (ii).   29. The method of claim 28, wherein 5%, 10%, 15%, or 20% of the cells that exhibit the highest luciferase activity in step (iii) comprise cells that exhibit the highest expression of the protein of interest.   30. The method of claim 28 or 29, wherein the luciferase activity is measured by a Bright-Glo luciferase assay on a Centro LB 960 photometer during an acquisition time of 5 seconds.   The luciferase activity of at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 cells is examined in step (iii), 28- 30. The method according to any one of 30.   (Iv) further comprising the step of selecting from about 1% to about 20% of the cells examined in step (iii), wherein the selected cells are cells that exhibit the highest luciferase activity in step (iii) 32. A method according to any one of claims 28 to 31.   33. The method of claim 32, further comprising (v) examining the expression level of the protein of interest in the selected cells at the end of step (iv). A method of obtaining a cell line that expresses a protein of interest,
(I) screening cells according to the method of any one of claims 27 to 33;
(Ii) selecting a cell that exhibits the highest expression of the protein of interest; and (iii) establishing a cell line from the cell. A method for producing a protein of interest,
(I) culturing a cell line obtained according to the method of claim 34 under conditions capable of expressing the protein of interest;
(Ii) A method comprising the step of recovering the protein of interest.   36. The method of claim 35, further comprising the step of purifying the protein of interest.   40. The method of claim 36, further comprising the step of formulating the protein of interest into a pharmaceutical composition. A method for producing the protein according to any one of claims 1 to 8,
(I) culturing the cell according to any one of claims 18 to 22 under conditions capable of expressing the protein according to any one of claims 1 to 8;
(Ii) A method comprising the step of recovering the protein according to any one of claims 1 to 8.   40. The method of claim 38, further comprising purifying the protein of any one of claims 1-8.

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