This application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing was created on Apr. 28, 2023, is named “23-0666-WO-US_SequenceListing_ST25.txt” and is 347,529 bytes in size.
The present invention relates to a monomeric streptavidin-expressing construct and a host cell into which a recombinant vector comprising the construct has been introduced.
Cancer is currently one of the diseases that cause the most deaths worldwide, and the incidence of cancer is continuously increasing due to an increase in average life expectancy and a decrease in the age of cancer onset. According to the 2013 statistical data provided by the Korean National Cancer Center, the total number of Korean cancer patients enrolled in the Cancer Registry Statistics Department in 2010 is 202,053 and the number of cancer patients has continued to increase.
Therefore, with respect to cancer, omnidirectional research on cancer treatment from cell-level basic research has been conducted worldwide, but the mechanism of cancer development still remains unclear, and it is difficult to prevent cancer recurrence and cure cancer. Thus, demand for anticancer drugs is explosively increasing, and enormous research funds are being invested in research institutes and companies. However, not only high cancer diagnosis and chemotherapy costs that are direct medical costs, but also indirect costs due to contraction of social and economic activities after cancer onset, rehabilitation, and patient care are additionally required, which cause a great economic burden for the families of cancer patients and all members of society. Thus, the introduction of low-cost new technology for cancer treatment is required.
Meanwhile, streptavidin and avidin proteins are proteins having a high binding affinity for biotin, and if their specific interaction with biotin is used, they may be applied to various biological applications, such as the use of anticancer drugs or immune cells that specifically target tumors expressing biotin.
However, the tetrameric form of these proteins may lead to unwanted cross-linking of the biotin conjugates, and thus it is required to develop monomeric streptavidin whose biotin binding activity is maintained. In recent years, monomeric avidin-like proteins have been developed and reported, such as monomeric rhizavidin developed by introducing a mutation into rhizavidin, which is an avidin-like protein, or monomeric proteins developed by fusing streptavidin and rhizavidin sequences. However, in order to use these monomeric avidin-like proteins in biological applications, it is necessary to obtain high-purity proteins with guaranteed solubility through purification processes. In addition, the avidin-like proteins have a problem in that they are rapidly degraded in serum when injected in vivo, which limits their use in clinical research.
Throughout the specification, a number of publications and patent documents are referred to and cited. The disclosure of the cited publications and patent documents is incorporated herein by reference in its entirety to more clearly describe the state of the related art and the present disclosure.
An object of the present invention is to provide a monomeric streptavidin (mSA)-expressing construct.
Another object of the present invention is to provide a recombinant vector comprising the construct and a host cell transformed therewith.
Still another object of the present invention is to provide a method for screening a regulatory gene for constructing a monomeric streptavidin (mSA)-expressing gene construct.
However, objects to be achieved by the present invention are not limited to the above-mentioned objects, and other problems not mentioned herein will be clearly understood by those skilled in the art from the following description.
Hereinafter, various embodiments described herein will be described with reference to figures. In the following description, numerous specific details are set forth, such as specific configurations, compositions, and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known processes and preparation techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase “in one embodiment” or “an embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.
Unless otherwise stated in the specification, all the scientific and technical terms used in the specification have the same meanings as commonly understood by those skilled in the technical field to which the present invention pertains.
According to one embodiment of the present invention, there is provided a gene construct comprising: a gene encoding monomeric streptavidin (mSA); a gene encoding maltose-binding protein (MBP); and a regulatory gene that regulates the expression of the gene encoding monomeric streptavidin.
As used herein, the term “gene construct” may refer to a construct which enables the expression of a protein of interest when cloned or introduced into a host strain or cell by transformation, and which comprises not only a gene encoding the protein of interest, but also an essential regulatory gene operably linked so that the gene encoding the protein of interest can be expressed.
As used herein, the term “regulation” or “regulation of expression” may mean that transcription and translation of a specific gene are activated or inhibited.
In the present invention, the “streptavidin” is a protein having a high binding affinity for biotin and has been applied to various biological applications due to its specific interaction with biotin. The amino acid sequence of the streptavidin protein may be represented by SEQ ID NO: 1, and the gene encoding the streptavidin may be represented by SEQ ID NO: 2, without being limited thereto.
In the present invention, the “monomeric streptavidin (mSA)” is a streptavidin that exists as a monomer so that the streptavidin may form a tetramer and cause unwanted cross-linking of biotin conjugate.
In the present invention, a gene encoding maltose-binding protein (MBP) may further be introduced into the host cell. The “maltose-binding protein (MBP)” is a part of the maltose/maltodextrin system of Escherichia coli, which is an about 42.5 kDa protein responsible for the uptake and efficient catabolism of maltodextrin. The maltose-binding protein (MBP) may be represented by the amino acid sequence of SEQ ID NO: 3, and the gene encoding the maltose binding protein may be represented by SEQ ID NO: 4, without being limited thereto.
In the present invention, the regulatory gene may refer to a nucleic acid fragment which structurally comprises a binding site for DNA-dependent RNA polymerase, transcription initiation sites and binding sites for transcription factors, repressor and activator protein binding sites, and any other sequences of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription, without being limited thereto.
In the present invention, the regulatory gene may be operably linked 5′ upstream of the initiation codon of the gene encoding the monomeric streptavidin.
In the present invention, the regulatory gene may be at least one selected from the group consisting of a ribosome binding site (RBS), a 5′-untranslated region (5′-UTR), a transcription factor binding site, and an inducible promoter, without being limited thereto.
In the present invention, the “ribosome-binding site (RBS)” is responsible for the recruitment of ribosomes upstream of the initiation codon of the gene to proceed with translation. The prokaryotic ribosome binding site contains a Shine-Dalgarno (SD) sequence having a 5′-AGGAGG-3′ sequence. The 3′ end of 16S rRNA complementarily binds to the Shine-Dalgarno sequence to initiate translation, and the complementary sequence CCUCCU is called the anti-Shine-Dalgarno (ASD) sequence.
In the present invention, the “5′-untranslated region (5′-UTR)” refers to untranslated regions flanking both sides of the 5′ coding region which is translated into amino acids of mRNA. It is considered a junk in the evolutionary process, but is known to play a major role in regulating gene expression.
In the present invention, the transcription factor binding site is a DNA region that serves to turn on or off a specific gene nearby. The transcription factor binding site may be at least one selected from the group consisting of a promoter, an enhancer, and a silencer of the gene encoding the regulatory protein, without being limited thereto.
In the present invention, the regulatory gene preferably causes the monomeric streptavidin to be expressed in the periplasm of the host cell when the recombinant vector comprising the gene construct is transformed into the host cell, because the utilization of the expressed monomeric streptavidin is higher than when the monomeric streptavidin remains inside the host cell or is released without remaining in the periplasm.
In the present invention, the regulatory gene may be one represented by any one of SEQ ID NOs: 26 to 91.
In the present invention, the regulatory gene may have a total Gibbs free energy change (ΔGtotal) of 0 or less. The “total Gibbs free energy change (ΔGtotal)” refers to the difference in Gibbs free energy between before and after an mRNA transcript of the regulatory gene binds to the 30S ribosomal subunit complex during the translation of the monomeric streptavidin. When the total Gibbs free energy change amount (ΔGtotal) is 0 or less, the transcription and translation ability of the gene encoding the monomeric streptavidin may increase. The total Gibbs free energy change (ΔGtotal) may be calculated using Equations 1 and 2 below.
ΔGtotal=(ΔGfinal)−(ΔGinitial) [Equation 1]
(ΔGfinal)−(ΔGinitial)=[(ΔGmRNA-rRNA)+(ΔGspacing)+(ΔGstacking)+(ΔGstandby)+(ΔGstart)]−(ΔGmRNA) [Equation 2]
In Equation 1 and Equation 2 above, “ΔGfinal” is the Gibbs free energy change after the 30S ribosomal subunit complex binds to an mRNA transcript of the regulatory gene, and “ΔGinitial” is the Gibbs free energy change before the 30S ribosomal subunit complex binds to the mRNA transcript of the regulatory gene. In addition, in Equation 2 above, “ΔGmRNA-rRNA” is the Gibbs free energy change when a reaction that forms a complex of the mRNA of the regulatory gene and the 30S ribosomal subunit occurs, “ΔGspacing” is a Gibbs free energy penalty that occurs when the spacing between the sequence forming the 30S ribosomal subunit complex and the initiation codon in the mRNA transcript of the regulatory gene is not optimized, “ΔGstacking” is the Gibbs free energy change of nucleotides stacked in the region of the spacing, “ΔGstandby” is the Gibbs free energy penalty when a binding reaction between the standby site of the mRNA transcript of the regulatory gene and a ribosome occurs, “ΔGstart” is the Gibbs free energy change when a reaction that forms an mRNA-tRNA complex occurs, and “ΔGmRNA” is the Gibbs free energy change when the mRNA transcript of the regulatory gene forms a folded complex structure.
In the present invention, each Gibbs free energy change (ΔG) may be calculated by software such as NUPACK, ViennaRNA, or UNAfold, which performs calculations in consideration of variables such as interaction of gene strands in a diluted solution, concentration, complexity of base pairing, and knot structure, without being limited thereto.
In the present invention, the regulatory gene may have a translation initiation rate (TIR) controlled within a specific range so as to maximize the production of the monomeric streptavidin.
In the present invention, the “translation initiation rate (TIR)” may be calculated using Equation 3 below, and is an important factor for gene expression because the translation step in synthetic biology is a step that limits the rate of total protein production.
TIR=exp[k{(ΔGtotal)−(ΔG1total)}] [Equation 3]
In Equation 3 above,
TIR is in units of AU;
k is the Boltzmann constant and may be 0.4 to 0.6 mol/kcal;
ΔGtotal is as defined in Equation 1 above; and
ΔG1total corresponds to the Gibbs free energy change in the gene construct of the present invention, which does not contain the regulatory gene, and preferably, may correspond to free energy change in the vector which does not contain the regulatory gene and in which the remaining sequences are the same, without being limited thereto. Thus, when the regulatory gene is not contained, the translation initiation rate corresponds to 1 AU.
In the present invention, the regulatory gene is preferably regulated so that the translation initiation rate is 50 to 45,000 AU, preferably 900 to 45,000 AU, because the transformed strain is capable of producing the monomeric streptavidin with high efficiency, without being limited thereto.
In the present invention, the sequence length of the regulatory gene may be 15 to 39 bp, preferably 26 to 31 bp, without being limited thereto.
In the present invention, the regulatory gene may comprise the gene sequence “AGG” represented by SEQ ID NO: 5, the regulatory gene may comprise the gene sequence “TAGG” represented by SEQ ID NO: 6, and the regulatory gene may comprise the gene sequence “ATAGG” represented by SEQ ID NO: 7, without being limited thereto.
In the present invention, the spacing between the 3′ end of the gene sequence represented by any one of SEQ ID NOs: 5 to 7 in the regulatory gene and the initiation codon may be 6 to 13 bp, preferably 6 to 10 bp. When the spacing is 6 to 13 bp, the Gibbs free energy penalty (ΔGspacing) for the unoptimized spacing between the sequence forming the rRNA complex and the initiation codon in the mRNA transcript may be minimized, resulting in an increase in the expression level of the monomeric streptavidin.
In a preferred example of the present invention, the regulatory gene may have a total Gibbs free energy change (ΔGtotal) of 0 or less as calculated by Equation 1 above, a translation initiation rate (TIR) of 900 to 45,000 AU, and a sequence length of 26 to 31 bp, and may comprise a gene sequence represented by any one of SEQ ID NOs: 5 to 7, and the spacing between the 3′ end of the gene represented by any one of SEQ ID NOs: 5 to 7 and the initiation codon of the gene encoding the monomeric streptavidin may be 6 to 10 bp.
In another preferred example of the present invention, the regulatory gene may be represented by SEQ ID NO: 32 or 36.
According to another embodiment of the present invention, there is provided a recombinant vector comprising the gene construct of the present invention.
In the present invention, the recombinant vector may be a constitutive expression vector or an inducible expression vector, and may be derived from, for example, at least one plasmid selected from among pKD13, pCP20, pMA1, pUC19, pJL, pBAD, pET, pGEX, pMAL, pALTER, pCal, pcDNA, pDUAL, pTrc, pQE, pTet, pProEX HT, pPROLar.A, pPROTet.E, pRSET, pSE280, pSE380, pSE420, pThioHis, pTriEx, pTrxFus, Split GFP Fold ‘n’ Glow, pACYCDuet-1, pCDF-1b, pCDFDuet-1, pCOLADuet-1, pLysS, pRSF-1b, pRSFDuet-1, pT7-FLAG, T7Select, pCMV, pBluescript, pBac, pAc, pFastBacHT, pFastBac, pAO815, pPIC, pESC, pCas9, pwtCas9-bacteria, pgRNA-bacteria, and pGRG plasmids, without being limited thereto.
In one example of the present invention, the pKD13 may be about 3.4 kbp in size, and may contain beta-lactamase, Tn5 neomycin phosphotransferase, lambda terminator, and R6K gamma replication origin genes.
In one example of the present invention, the pCP20 plasmid may be about 9.4 kbp in size, and may contain EcoRI, cat, Pstl, HindIII, Ci857, flp, bamHi, beta-lactamase, mobA, mob2, and repA101ts gene regions.
In one example of the present invention, the pMA1 plasmid may be derived from Microcystis aeruginosa f. aeruginosa Kutzing, may be about 2.3 kbp in size, and may contain a HincII gene region.
In one example of the present invention, the pJL plasmid may have an empty backbone and be based on an RNA virus.
In one example of the present invention, the pBAD, pCMV and pCMV plasmids may be expressed in mammalian host cells, contain a CMV and a promoter, and have ampicillin resistance.
In one example of the present invention, the pET, pBluescript, pCal and pcDNA plasmids may be expressed in bacterial host cells, contain a T7 or Lac promoter, and have ampicillin resistance.
In one example of the present invention, the pMAL and pGEX plasmids may be expressed in bacterial host cells, contain a Tac promoter, and have ampicillin resistance.
In one example of the present invention, the pALTER plasmid may be expressed in bacterial host cells, contain a T7 promoter, and have tetracycline resistance.
In one example of the present invention, the pDUAL plasmid may be expressed in bacterial host cells, contain a T7 or Lac promoter, and have kanamycin resistance.
In one example of the present invention, the pTrc plasmid may be expressed in bacterial host cells, contain a trc promoter, and have ampicillin resistance.
In one example of the present invention, the pUC19 plasmid is a vector that is expressed in bacterial host cells, comprises about 2.6-kbp circular double-stranded DNA, and has an MCS region opposite to that of pUC18. The pU19 vector is most widely used for transformation, and host cells into which foreign DNA has been introduced by the pU19 may be distinguished because the color of colonies in a growth medium is different from that of a control group.
In one example of the present invention, the pQE plasmid may contain a T5-lac promoter and have ampicillin resistance.
In one example of the present invention, the pTet plasmid contains a CMV promoter under the control of a regulatory sequence from the tet operon, and thus when cells are co-transfected with the pTet plasmid and the transactivator pTet-tTAk, they may express a protein only in the absence of doxycycline.
In one example of the present invention, the pCas9, pwtCas9-bacteria and pgRNA-bacteria plasmids may be used to express the Cas9 nuclease gRNA using CRISPR technology.
According to still another embodiment of the present invention, there is provided a transformed host cell into which the recombinant vector of the present invention has been introduced.
In the present invention, the method of transforming the host cells may be performed according to a conventional introduction method known in the art, and is not particularly limited to any specific method, but examples thereof include a bacterial transformation method, a CaCl2) precipitation method, a Hanahan method with improved efficiency using dimethyl sulfoxide (DMSO) as a reducing agent in the CaCl2) method, an electroporation method, a calcium phosphate precipitation method, a protoplast fusion method, an agitation method using silicon carbide fibers, an agrobacterium-mediated transformation method, a transformation method using PEG, a dextran sulfate-mediated transformation method, a lipofectamine-mediated transformation method, and a desiccation/inhibition-mediated transformation method.
In the present invention, when the host cells are administered to a subject having cancer, the monomeric streptavidin may be effectively expressed only in cancer tissue. Thus, when the host cells of the present invention are administered to a subject having cancer, the viability thereof is preferably lower in normal tissue than in cancer tissue, because there is no infection in the normal tissue and the monomeric streptavidin may be expressed only in the cancer tissue. Here, the normal tissue may be a tissue of an organ selected from the group consisting of lung, liver, and spleen, without being limited thereto.
In addition, in the present invention, the host cell may include cells of mammalian, plant, insect, fungal or cellular origin. For example, the host cell may be of at least one type selected from the group consisting of bacterial cells such as Escherichia coli, Streptomyces or Salmonella sp. strains; yeast cells; fungal cells such as Pichia pastoris; insect cells such as Drosophila or Spodoptera Sf9 cells; animal cells such as Chinese hamster ovary (CHO) cells, SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T cells, bow melanoma cells, HT-1080 cells, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, or PERC.6 cells (human retinal cells; and plant cells, without being limited thereto.
In one example of the present invention, the host cell may be a bacterial cell, preferably an anaerobic strain, and in this case, when the host cell is injected into the human body for the purpose of cancer diagnosis and treatment, it targets the inside of cancer tissue, an environment which is deficient in oxygen due to incomplete blood vessel formation. Thus, when a recombinant vector comprising a reporter protein that may be imaged in real time and an anticancer protein is introduced into this strain so that they may be simultaneously expressed in a balanced manner, it is possible not only to monitor in real time the location of the strain or the cancer tissue pre-targeted by the strain by using a biotinylated diagnostic agent, but also to increase the cancer targeting efficiency of a biotinylated anticancer agent, thereby diagnosing and treating cancer very effectively.
In one example of the present invention, the bacteria may be at least one selected from the group consisting of Salmonella sp. strains, Clostridium sp. strains, Bifidobacterium sp. strains, and E. coli sp. strains, and more preferably, may be at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, and Salmonella enteritidis, and even more preferably, may be Salmonella typhimurium, without being limited thereto.
In the present invention, the “Salmonella typhimurium” is a Salmonella sp. bacterium that causes typhoid fever. The Salmonella typhimurium is a rod-shaped bacillus that has a flagellum and is Gram-negative. The Salmonella typhimurium is weak to heat and dies within 20 minutes at 60° C. Also, the Salmonella typhimurium may cause salmonellosis, a kind of food poisoning, through primary contamination from livestock, wild animals, carriers, milk, eggs or the like and also by salads which are susceptible to secondary infection from contaminated meat, etc.
In the present invention, the “Salmonella choleraesuis” is a well-known Salmonella sp. bacterium that causes hog cholera and infects both humans and animals. The Salmonella choleraesuis is a major Salmonella sp. bacterium that causes acute sepsis. This bacterium is a Gram-negative facultative anaerobic bacillus that has peritrichous flagella and is motile. This bacterium is distinguished from Escherichia coli in that it is not able to decompose lactose, does not form indole, and does not produce hydrogen sulfide. This bacterium optimally grows at a temperature of 35 to 37° C., is capable of proliferating at a temperature of 10 to 43° C., and is killed by heating at 60° C. for 20 minutes. This bacterium optimally grows at a pH of 7.2 to 7.4 and is 0.5 to 0.8×3 to 4 μm in size.
In the present invention, the “Salmonella enteritidis” is a Salmonella sp. bacterium that causes bacterial infection-type food poisoning, and is also called Bacillus enteritidis. The Salmonella enteritidis is a representative bacterium of the genus Salmonella, which may infect all animals and has a very high host adaptability. This bacterium is a Gram-negative, facultative anaerobic bacillus that has peritrichous flagella and is motile. This bacterium is distinguished from Escherichia coli in that it is not able to decompose lactose, does not form indole, and does not produce hydrogen sulfide. This bacterium optimally grows at a temperature of 35 to 37° C., is capable of proliferating at a temperature of 10 to 43° C., and is killed by heating at 60° C. for 20 minutes. It optimally grows at a pH of 7.2 to 7.4 and is 0.5 to 0.8×3 to 4 μm in size.
In the present invention, the “Salmonella infantis” is a strain that causes infection by eggs or poultry meat, and the Salmonella paratyphi and the Salmonella typhi are strains that cause typhoid fever.
In the present invention, the bacteria may be attenuated so that it may exhibit reduced virulence and other side effects when administered to a subject.
In one example of the present invention, the bacteria may express a modified form of a gene encoding at least one selected from the group consisting of aroA, aroC, aroD, aroE, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, sipC, clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA, spoA, and spoT.
In another example of the present invention, the bacteria may be attenuated due to lack of guanosine polyphosphate synthesis ability. The guanosine polyphosphate may be guanosine-5-diphosphate-3-diphosphate (ppGpp), and the host cells may lack the ability to synthesize guanosine-5-diphosphate-3-diphosphate (ppGpp), due to modification of a gene encoding either relA that hydrolyzes guanosine-5-diphosphate-3-diphosphate (ppGpp) or spot that synthesizes guanosine-5-diphosphate-3-diphosphate (ppGpp), without being limited thereto.
In the present invention, the method of modifying the gene in the bacteria may be performed by a method of deleting or disrupting various genes known in the art. For example, the method of deleting and disrupting genes may be performed by a method such as homologous recombination, chemical mutagenesis, irradiation mutagenesis, or transposon mutagenesis, without being limited thereto.
According to another embodiment of the present invention, there is provided a method for screening a regulatory gene for regulating the expression of monomeric streptavidin (mSA), the method comprising steps of: introducing a gene encoding monomeric streptavidin (mSA) and a candidate regulatory gene into a vector; and measuring the expression level of the gene encoding monomeric streptavidin expressed by the vector.
In the present invention, the candidate regulatory gene may satisfy at least one of the following conditions, but is not limited thereto: the candidate regulatory gene has a total Gibbs free energy change (AGtotal) controlled to 0 or less; the translation initiation rate of the candidate regulatory gene is in the range of 900 to 9,000 AU; the sequence length of the candidate regulatory gene is 15 to 39 bp; the candidate regulatory gene comprises a gene sequence represented by any one of SEQ ID NOs: 5 to 7; and the spacing between the 3′ end of the gene sequence and the initiation codon is 6 to 13 bp.
In the present invention, a gene encoding maltose-binding protein (MBP) may be further introduced into the vector in the step of introducing.
In the present invention, the step of measuring the expression level may be performed by measuring the expression level of monomeric streptavidin expressed from a host cell transformed with the vector into which the gene encoding monomorphic streptavidin and the candidate regulatory gene have been introduced.
In the present invention, when the measured expression level of monomeric streptavidin is higher than that before the candidate regulatory gene is introduced, it may be determined that the candidate regulatory gene is a gene that increases the expression of monomeric streptavidin.
In addition, in the present invention, the step of measuring the expression level may be performed by measuring the expression level of monomeric streptavidin expressed in the periplasm of the transformed host cell.
In the present invention, when the monomeric streptavidin is expressed in the periplasm of the transformed host cell, it may be determined that the candidate regulatory gene is a gene that increases the expression of the monomeric streptavidin. In the present invention, when the monomeric streptavidin is expressed in the periplasm of the transformed host cell as described above, the utilization of the expressed monomeric streptavidin may be higher than when the monomeric streptavidin is expressed inside the host cell or when the monomeric streptavidin is released without remaining in the periplasm after expression.
In the present invention, the step of measuring the expression level may further comprise a step of culturing the transformed host cell.
In the present invention, the step of culturing may be performed using an LB (Lysogeny broth) medium containing antibiotics. The LB medium is one developed by Giuseppe Bertani to optimize the growth and plaque formation of Shigella sp. strains, and generally contains peptides, casein peptone, vitamins (including vitamin B), trace elements (nitrogen, sulfur, and magnesium), and minerals, and the osmotic pressure thereof may be controlled by sodium chloride, without being limited thereto.
In the screening method of the present invention, contents regarding the total Gibbs free energy change (ΔGtotal), the translation initiation rate, the host cell, and introduction overlap with those described above, and thus detailed description thereof will be omitted below to avoid excessive complexity of the specification.
When the recombinant vector comprising the gene construct according to the present invention is introduced into host cells, the monomeric streptavidin may be expressed with high productivity in the host cells.
In addition, according to the present invention, when host cells expressing monomeric streptavidin are administered to a subject having a tumor, the monomeric streptavidin expressed from the host cells may maintain its functionality in vivo. Therefore, when a biotinylated drug for diagnosing or treating a tumor is administered together with the tumor-targeting host cells, the biotinylated drug may bind to monomeric streptavidin and selectively act only on cancer tissue.
Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.
In order to construct a plasmid for mSA expression in a recombinant strain, the monomeric streptavidin (mSA) gene represented by SEQ ID NO: 2 was synthesized (Macrogen, Korea), amplified, digested with restriction enzymes EcoRI and SalI, and purified to obtain a gene amplification product which was then cloned into a pBAD24 plasmid digested with the same restriction enzymes, thus constructing a pBAD-mSA (B-mSA) plasmid.
Additionally, in order to increase the expression of the mSA gene, BBa_B0032, BBa_B0030, and BBa_B0034, which are the ribosome binding sites (RBSs) shown in Table 1 below, were each inserted downstream of the promoter, thereby constructing pBAD_RBS 0.3-mSA (B_R0.3-mSA), pBAD_RBS 0.6-mSA (B_R0.6-mSA), and pBAD_RBS 1.0-mSA (B_R1.0-mSA) plasmids.
In addition, in order to increase the expression and solubility of the gene, the mSA gene was amplified using the pBAD-mSA plasmid as a template, and then digested with restriction enzymes EcoRI and HindIII and purified to obtain a gene amplification product which was then cloned into each of pMA1_p2x and pMA1_c2x plasmids digested with the same restriction enzymes, thereby constructing pMA1_p2x-mSA (M_p-mSA) and pMA1_c2x-mSA (M_c-mSA) plasmids.
Next, for use in animal experiments, the maltose binding protein (MBP)-encoding gene represented by SEQ ID NO: 4, the mSA gene represented by SEQ ID NO: 2, and the BBa_B0034 sequence were each cloned into a pBAD24 plasmid, thereby constructing pBAD_p2x-mSA (B_p-mSA), pBAD c2x-mSA (B_c-mSA), pBAD_RBS 1-p2x-mSA (B_R1.0-p-mSA), and pBAD_RBS1-c2x-mSA (B_R1.0-c-mSA) plasmids.
In order to increase the expression level and functionality of mSA, gene constructs in which the existing RBS was substituted with a new regulatory gene were additionally constructed (Table 1 below). First, a sequence library was prepared by analyzing the RBS sequence of the plasmid. Next, the translation initiation rate (TIR) of the B_p-mSA plasmid was analyzed using the RBS calculator (Penn State University) program, and then a regulatory gene library having a translation initiation rate value ranging from 3.97 to 42,889 as calculated by the RBS library calculator was constructed. The regulatory gene constructed according to the library was cloned to substitute for the RBS sequence of the B_p-mSA plasmid, and then the resulting colonies were selected, thereby constructing the final plasmids pBAD_R01-p2x-mSA (B_R01-p-mSA), pBAD_R02-p2x-mSA (B_R02-p-mSA), pBAD_R1-p2x-mSA (B_R1-p-mSA), pBAD_R11-p2x-mSA (B_R11-p-mSA), pBAD_R12-p2x-mSA (B_R12-p-mSA), pBAD_R13-p2x-mSA (B_R13-p-mSA), pBAD_R2-p2x-mSA (B_R2-p-mSA), and pBAD_R21-p2x-mSA (B_R21-p-mSA) (see Table 1 below).
The name, abbreviation, backbone plasmid and entire sequence of each gene construct obtained in Examples 1-1 to 1-3 are shown in Table 1 below.
For the sequence from the promoter to the initiation codon of the ribosome binding site (RBS) S) constructed in Example 1-3, in order to confirm the mSA expression ability of the gene construct depending on the total Gibbs free energy change (ΔGtotal), the total Gibbs free energy change (ΔGtotal) was calculated by calculating the following parameters, and the results are shown in Table 2 below: ΔGmRNA-rRNA which is the Gibbs free energy change when a reaction that forms a complex of the mRNA of the regulatory gene and the 30S ribosomal subunit occurs; ΔGspacing which is a Gibbs free energy penalty that occurs when the spacing between the sequence forming the 30S ribosomal subunit complex and the initiation codon in the mRNA transcript of the regulatory gene is not optimized; ΔGstacking which is the Gibbs free energy change of nucleotides stacked in the region of the spacing; ΔGstandby which is the Gibbs free energy penalty when a binding reaction between the standby site of the mRNA transcript of the regulatory gene and a ribosome occurs; ΔGstart which is the Gibbs free energy change when a reaction that forms an mRNA-tRNA complex occurs; and ΔGmRNA which is the Gibbs free energy change when the mRNA transcript of the regulatory gene forms a folded complex structure. Here, the total Gibbs free energy change (ΔGtotal) was calculated using Equations 1 and 2 below.
ΔGtotal=(ΔGfinal)−(ΔGinitial) [Equation 1]
(ΔGfinal)−(ΔGinitial)=[(ΔGmRNA-rRNA)+(ΔGspacing)+(ΔGstacking)+(ΔGstandby)+(ΔGstart)]−(ΔGmRNA) [Equation 2]
In order to confirm the mSA expression ability of the plasmid depending on the translation initiation rate (TIR) of the regulatory gene, the translation initiation rate of each regulatory gene sequence constructed as described above was calculated, and the results are shown in Table 3.
As shown in Table 3, it was confirmed that, among the regulatory genes of the constructed plasmids, the regulatory genes of SEQ ID NOs: 29 to 37 and 65 to 91 had translation initiation rates in the range of 50 to 45,000 AU, and thereamong, the regulatory genes of SEQ ID NOs: 32 and 36 had translation initiation rates in the range of 900 to 9,000 AU.
In order to examine the mSA expression ability of the plasmid depending on whether not the regulatory gene sequence comprises the AGG, TAGG or ATAGG sequence and on the spacing between the 3′ end of the AGG sequence and the initiation codon, the regulatory gene sequence of each plasmid and the spacing (unit: bp) between the 3′ end of the AGG sequence and the initiation codon were analyzed, and the results are shown in Table 4 below.
As shown in Table 4, it was confirmed that, among the regulatory genes of the constructed plasmids, the regulatory genes of SEQ ID NOs: 26, 27, 28, 29, 31, 32, 36, 37, 66, 67, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 and 91 contained the AGG sequence, and in particular, the spacing between the 3′ end of the AGG sequence in the regulatory genes of SEQ ID NOs: 32 and 36 and the initiation codon was 6 to 13 bp. In addition, it was confirmed that, among the regulatory genes of the constructed plasmids, the regulatory genes of SEQ ID NOs: 32, 36, 66, 76, 77, 78, 82, 84, 86, 87, 88, 89, 90 and 91 contained the TAGG or ATAGG sequence.
After each of the plasmids constructed in Example 1 was transformed into Escherichia coli (DH5a, or MG1655), or Salmonella sp. strains (SHJ2037), each of the transformed strains was cultured overnight using an LB solid medium containing ampicillin. Then, the resulting colonies were diluted at a ratio of 1:100 using an LB liquid medium containing antibiotics, and when the OD600 value reached 0.5 to 0.7 during additional culture, arabinose was added to the culture at a final concentration of 0.1%, followed by culturing in a shaking incubator under conditions of 200 rpm and 37° C.
In order to analyze the expression level of the plasmid into which the mSA gene was inserted alone, recombinant E. coli colonies containing each of the plasmids B-mSA, B_R0.3-mSA, B_R0.6-mSA and B_R1.0-mSA constructed in Example 1 were transformed and cultured as described in Example 2. Next, the cultured recombinant E. coli was added to SDS-PAGE sample buffer based on OD4, boiled at 95° C. for 10 minutes, and then loaded on SDS-PAGE to determine the expression level of the protein, and the results are shown in
As shown in
The present inventors examined the mSA expression level of a strain transformed with an MBP-mSA plasmid in which the MBP gene was fused with mSA in order to increase the expression and solubility of mSA. Specifically, M_p-mSA and M_c-mSA plasmids obtained by fusion with the MBP gene were constructed as described in Example 1, and transformation and culture were performed as described in Example 2. When the OD600 value reached 0.5 to 0.7 during culture, isopropyl beta-D-1-thiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.1 mM, followed by culturing in a shaking incubator under conditions of 200 rpm and 37° C. The cultured recombinant E. coli was added to SDS-PAGE sample buffer based on OD4, boiled at 95° C. for 10 minutes, and then loaded on SDS-PAGE to confirm the expression level of the protein, and the results are shown in
As a result, as shown in
Western blot analysis was performed to analyze the mSA expression level and biotin binding activity of the recombinant strain transformed with the MBP-mSA plasmid. Specifically, the culture of the strain of Example 2 was diluted with PBS to 4×107 CFU/ml, and the pellet was collected by centrifugation at 13,000 rpm for 5 minutes. The pellet fraction was washed with PBS and mixed with SDS sample buffer containing 0.2% beta-mercaptoethanol (catalog number: EBA-1052, ELPIS BIOTECH) to obtain a strain lysate. Then, the strain lysate was electrophoresed on 12% SDS-PAGE gel, and the protein was transferred from the gel to a nitrocellulose membrane, followed by blocking with 5% skim milk at room temperature. Then, the expression level of mSA was confirmed using his tag antibody, and the biotin-binding activity of mSA was confirmed using biotinylated peroxidase. The results are shown in
As shown in
In addition, it could be confirmed that, although the expression level of the protein expressed from the M_c-mSA plasmid was higher than that from M_p-mSA, the biotin binding activity of MBP-fused mSA with the secretion sequence was higher.
In order to analyze the biotin binding activity of the recombinant strain transformed with the MBP-mSA plasmid, biotin uptake assay was performed, and the results are shown in
As a result, as shown in
In order to actually image the binding of the biotinylated fluorescent dye to the recombinant strain, the recombinant strains were fixed to slides and observed with a confocal microscope, and the results are shown in
As shown in the biotin uptake assay results in
SDS-PAGE was performed to examine the mSA expression and activity of the recombinant strain transformed with the RBS-added plasmid. Specifically, SD S-PAGE was performed on recombinant strains transformed with each of B_p-mSA and B_c-mSA plasmids obtained by cloning the MBP-mSA gene into the pBAD plasmid, and B_R1.0-p-mSA and B_R1.0-c-mSA plasmids obtained by adding the BBa_B0034 sequence to improve the expression of the plasmids, and the results are shown in
As shown in
Western blot analysis was performed to examine the mSA expression and activity of the recombinant strain transformed with the RBS-added plasmid. Western blot analysis was performed on B_p-mSA and B_c-mSA plasmids obtained by cloning the MBP-mSA gene into the pBAD plasmid, and B_R1.0-p-mSA and B_R1.0-c-mSA plasmids obtained by adding the BBa_B0034 sequence to improve the expression of the plasmids, in the same manner as in Experimental Example 2-2, and the results are shown in
As shown in
The present inventors analyzed the RBS sequence of the B_p-mSA plasmid to induce increased functional expression of the gene in the recombinant strain, and constructed B_R01-p-mSA, B_R02-p-mSA, B_R1-p-mSA, B_R11-p-mSA, B_R12-p-mSA, B_R13-p-mSA B_R2-p-mSA and B_R21-p-mSA plasmids as described in Example 1. A strain was transformed with each of the constructed plasmids and cultured. In order to examine the protein expression level of each of the recombinant strains, Western blot analysis was performed in the same manner as in Experimental Example 2-2, and the results are shown in
As shown in
Additional experiments were performed on the two selected strains transformed with each of the B_R1-p-mSA and B_R2-p-mSA plasmids having high mSA expression levels, and the results are shown in
As shown in
In addition, it was shown that the secretion level versus expression level of the protein was lower in the recombinant strains containing each of the B_R1-p-mSA and B_R2-p-mSA plasmids than in the recombinant strain containing the M_p-mSA plasmid, indicating that mSA expressed from each of the B_R1-p-mSA and B_R2-p-mSA plasmids remained in the periplasm of the strain. The biotin binding activity was higher in the order of the recombinant strains containing the BAD-mSA, B_R1-p-mSA, M_p-mSA and B_R2-p-mSA plasmids, respectively, and the secreted protein binding activity was higher in the order of the recombinant strains containing the M_p-mSA, BAD-mSA, B_R1-p-mSA, B_R2-p-mSA plasmids, respectively.
In addition, in order to analyze the biotin binding activity of the recombinant strain with improved expression, biotin uptake assay was performed in the same manner as in Experimental Example 2, and the results are shown in
As shown in
In order to actually image the binding of the biotinylated fluorescent dye to the recombinant strain, the cultured strains were fixed to slides and observed with a confocal microscope, and the results are shown in
As shown in
In order to confirm whether the mSA gene expressed in the constructed recombinant strain of the present invention is specific to biotin, as described in Experimental Example 1, each of the pBAD, B-mSA, BAD-mSA, B_R1-p-mSA and B_R2-p-mSA plasmids was transformed into Salmonella strains which were then cultured. Next, biotin uptake assay was performed in the same manner as in Experimental Example 2, and the results are shown in
As shown in
In order to confirm the biotin binding activity of the recombinant strain of the present invention, in vivo imaging system (IVIS) imaging was performed. Specifically, first, the CT26 cell line was subcutaneously injected into the flanks of Balb/c mice to construct tumor animal models. After 3 days form each recombinant strain was injected into the tumor animal model, biotinylated fluorescent dye was injected into each mouse. The results of IVIS imaging performed 6 hours after biotinylated fluorescent dye injection are shown in
As shown in
In addition, in order to confirm the biotin binding activity of the recombinant strain of the present invention, cancer tissue was harvested from the tumor animal model and imaged with an in vivo imaging system (IVIS). Specifically, 24 hours after the biotinylated fluorescent dye was injected into the tumor animal model, the tumor was harvested from each group and imaged with an IVIS to detect the signal of the biotinylated fluorescent dye, and the results are shown in
As shown in
In order to confirm the multiple-biotin-binding activity of the recombinant strain of the present invention, in vivo imaging system (IVIS) imaging was performed. Specifically, first, the CT26 cell line was subcutaneously injected into the flanks of Balb/c mice to construct tumor animal models. The recombinant strain was injected into the tumor animal models. Three days after injecting the recombinant strain into the tumor animal models, the biotinylated fluorescent dye was injected (first injection). Two days later, the biotinylated fluorescent dye was injected into the same tumor animal models (second injection). IVIS imaging was performed before, 6 hours after, and 9 hours after the first injection of the fluorescent dye, and then IVIS imaging was performed before, 6 hours after, and 9 hours after the second injection of the fluorescent dye, and the results are shown in
As shown in
Specifically, through the above experiments, it was confirmed that, when the recombinant vector or construct according to the present invention, especially the regulatory gene according to the present invention, is included, the monomeric streptavidin (mSA) expressed has excellent stability and can strongly bind to external biotin, and this is effective even in vivo, and treatment with the biotinylated fluorescent dye may be performed multiple times or at adjusted time intervals.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only description of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.
Number | Date | Country | Kind |
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10-2020-0143486 | Oct 2020 | KR | national |
This application is a U.S. National Stage entry of International Patent Application no. PCT/KR2021/015406, filed Oct. 29, 2021, which claims the benefit of priority of Korean Patent Application no. 10-2020-0143486, filed Oct. 30, 2020.
Filing Document | Filing Date | Country | Kind |
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PCT/KR2021/015406 | 10/29/2021 | WO |