Improved process for the preparation of recombinant lectin proteins

Abstract

The present invention relates to a process for preparing a recombinant lectin having the amino acid sequence of SEQ ID NO: 1, wherein the process comprises fed-batch fermentation of a clone in a host cell at a carbon to nitrogen ratio of 3:1 to 6:1 and at a specific feed rate. The present invention also relates to a purification process for the recombinant lectin having the amino acid sequence of SEQ ID NO: 1.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of Indian Provisional Application No. 201821008382, filed on September 7, 2018, and Indian Provisional Application No. 201921001559, filed on January 14, 2019, the entire contents of which are incorporated herein by reference.

[0002] Field of the Invention The present invention relates to a recombinant lectin protein and a method for preparing a recombinant lectin protein. The present invention also relates to a method for purifying a crude recombinant lectin protein.

[0003] Background of the Invention Lectins are sugar - binding proteins, which are macromolecules highly specific for the sugar moieties of other molecules. Lectins recognize at the cellular and molecular levels and play numerous roles in biological recognition phenomena involving cells, sugar chains, and proteins. They are divalent or multivalent sugar - binding proteins that bind to glycoproteins and precipitate them, and agglutinate red blood cells. Lectins found in animals have been found to often assist in cell - cell interactions, while plant lectins are known to defend against potential predators or pathogens. Some lectins can detect cancer - related glycans and, therefore, have the potential to serve as biomarkers for malignancies and support the study of changes in glycosylation motifs in cancer cell lines.

[0004] Purified lectins are important in clinical practice because they are also used in blood typing. Some glycolipids and glycoproteins on an individual's red blood cells can be identified by lectins. Many lectins are used as biomarkers for the early detection of malignant growth or as autophagy inducers, while others also exhibit the ability to suppress cancer growth through apoptosis. Due to uncontrolled cell proliferation, some glycan portions are expressed as antigens on cancer cells. Lectins are used as drug delivery agents in cancer therapy because they specifically bind to malignant tumors. Furthermore, lectins also modulate cancer-related pathways, so they have potential as diagnostic and therapeutic agents for cancer.

[0005] There are several antigens to which lectins bind, and which are characterized on the surface of cancer cells; most antigens are specific to certain types of cancer, and lectin binding to these antigens can suppress cancer growth through the induction of apoptosis in cancer cells. Currently, most commercially available lectins are derived from plants and other eukaryotes.

[0006] Sclerotium rolfsii lectin (SRL) is a lectin isolated from the sclerotial bodies of the soil-derived plant pathogenic fungus S. rolfsii. SRL exhibits specificity for Thomsen-Friedenreich (TF) antigen and Tn antigen. The TF antigen is a disaccharide (Galβ1→3GalNAc-α-Ser / Thr) overexpressed on the cell surface of various human cancer cells. The Tn antigen is a monosaccharide (GalNAc-α-). Due to its specificity for the TF and Tn antigens, SRL has been shown to bind to human colorectal cancer, ovarian cancer, and leukemia cells. The crystal structure of SRL has been determined (Leonidas et al., J Mol Biol. 2007 May 11;368(4):1145-61).

[0007] While lectins offer many advantages as anti-cancer tools, they still suffer from numerous limitations, including a lack of selectivity, inconsistent quality and performance, and difficulty in easily scaling up production. Furthermore, plant-derived lectins are often reported to bind to a range of diverse glycan structures, thus lacking the selectivity required for many applications. Additionally, batch-to-batch variability is common when using plant lectins. Product quality depends on the isolation method of the plant material and the quality of the starting plant material itself.

[0008] Isolating lectins from natural sources is unreliable because the lectins obtained in this way lack consistency regarding the desired properties. Further isolation of proteins from natural sources is a costly and difficult process. The techniques used to isolate natural lectins usually result in very low yields, especially when the protein is present only at low concentrations. Furthermore, it is sometimes impossible to distinguish between isoforms of the same lectin. Therefore, they are obtained as mixtures, which introduces much uncertainty. In this sense, the production of recombinant lectins using recombinant DNA technology offers the advantage of providing a single protein with precise characteristics, in a dramatically shorter time, with better and more consistent yields, and at the same time, it is easily scalable. By using rDNA technology, the gene producing the protein of interest can be introduced into a suitable host. The protein can then be produced and isolated in a shorter time and with less effort compared to traditional methods.

[0009] International Publication No. 2010 / 095143 discloses recombinant lectin mutants Rec-2 and Rec-3, derived from the natural SRL sequence by substitutions of 3 or 5 amino acids, respectively. The crystal structures of these mutants have been reported (Peppa et al., Molecules. 2015 Jun 12;20(6):10848-65).

[0010] International Publication No. 2014 / 203261 discloses recombinant lectin variants derived from natural SRL sequences by 12 amino acid substitutions.

[0011] Indian patent application No. 350 / MUM / 2009 (PCT application, International Publication No. 2010 / 095143) is the first to disclose the amino acid sequence of Sequence ID No. 1 (referred to as Sequence ID No. 2 in No. 350 / MUM / 2009), derived from the natural amino acid sequence of Sequence ID No. 2 (referred to as Sequence ID No. 1 in No. 350 / MUM / 2009), which is derived from the fungus Sclerotium rolfsii. International Publication No. 2010 / 095143 describes the laboratory-scale production of recombinant lectin in E. coli cells, where the gene encoding the recombinant lectin is cloned into an expression vector. However, this method suffers from some drawbacks, including difficulty in controlling culture parameters such as dissolved oxygen and pH, and the inability to control protein expression. Furthermore, the culture medium produces few cell clumps, resulting in an overall low yield of purified protein. Moreover, the process cannot be industrially scaled up.

[0012] Lectins with the amino acid sequence of SEQ ID NO: 1 exhibit higher stability and solubility compared to natural lectins derived from the fungus Sclerotium rolfsii, and demonstrate cancer cell binding properties. Lectins have various applications in research, pharmaceutical, and biochemical technologies. For example, lectins with the amino acid sequence of SEQ ID NO: 1 have high pharmaceutical potential. Therefore, there is a need to devise processes to produce recombinant lectins that are higher yield, more cost-effective, easier to scale up, and / or have improved efficacy.

[0013] This invention was conceived with these problems in mind.

[0014] Object of the invention The main objective of the present invention is to overcome the shortcomings of prior art objects and to devise a highly efficient process for preparing the amino acid sequence of SEQ ID NO: 1.

[0015] Another object of the present invention is to provide a process that is high-yielding, cost-effective, and can be carried out using readily available raw materials.

[0016] Another object of the present invention is to provide an easily scalable process for preparing the amino acid sequence of SEQ ID NO: 1.

[0017] Another object of the present invention is to provide a process for producing the amino acid sequence of SEQ ID NO: 1 in a very pure form.

[0018] Summary of the Invention In one aspect of the present invention, a method for preparing recombinant lectin proteins is provided, comprising the step of expressing recombinant lectin proteins encoded by recombinant lectin genes in a host cell culture medium, wherein the cell expression is carried out under conditions such that the cells have a doubling time of 160 minutes or less. In some embodiments, the cell expression is carried out under conditions such that the cells have a doubling time of at least 100 minutes.

[0019] In one embodiment, the method includes expressing a recombinant lectin protein encoded by a recombinant lectin gene in a host cell culture medium at a temperature of 22°C or lower. In a further embodiment, the expression is carried out at a temperature of at least 15°C and 22°C or lower.

[0020] In some embodiments, the host cell for recombinant lectin expression is Escherichia coli (E. coli). In some embodiments, the recombinant lectin protein is selected from an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% homology to Sequence ID No. 1, Sequence ID No. 3, Sequence ID No. 4, or any of these sequences.

[0021] In some embodiments, the host cell culture medium has a volume of at least 10 L.

[0022] In some embodiments, the method includes a step of culturing host cells, the culturing including a growth period (during which the host cells are grown before protein expression) and an expression period (during which protein expression takes place). In further embodiments, the growth period is carried out at a higher temperature than the temperature at which the expression period takes place. In yet another embodiment, the temperature is lowered from the growth period to the expression period over a period of at least 4 hours and no more than 7 hours.

[0023] In some embodiments, the expression period is induced by the addition of an inducer at a concentration of at least 0.1 mM and 0.7 mM or less. In other embodiments, the inducer is added to the culture medium at a concentration of 0.5 mM or less. In yet another embodiment, the expression period is induced by the addition of an inducer when the absorbance of the culture medium is at least 25 and 40 or less.

[0024] In some embodiments, the method includes an expression period induced by the addition of an inducer, where the absorbance of the culture medium is at least 25 and 40 or less, and the inducer is at a concentration of at least 0.1 mM and 0.7 mM or less.

[0025] In another aspect of the present invention, a method for culturing host cells is provided, wherein the expression period is induced by the addition of an inducer at a concentration of at least 0.1 mM and no more than 0.7 mM, and the cell expression is carried out under conditions such that the cells have a doubling time of 160 minutes or less. In some embodiments, the inducer is added to the culture medium at a concentration of 0.5 mM or less.

[0026] In a further aspect of the present invention, a method for culturing host cells is provided, wherein the expression period is induced by the addition of an inducing substance when the absorbance of the culture medium is at least 25 and 40 or less, and the cell expression is carried out under conditions such that the cells have a doubling time of 160 minutes or less.

[0027] In some embodiments, the recombinant lectin protein is selected from amino acid sequences having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% homology to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or any of these sequences.

[0028] In certain embodiments, the absorbance is a measurement of the host cell population in the culture medium and is measured at 600 nm.

[0029] In some embodiments, the inducer is IPTG.

[0030] The present invention also provides a method for preparing a recombinant lectin protein, the method comprising the step of expressing the recombinant lectin protein encoded by the recombinant lectin gene in a host cell culture, wherein the expression of the cells is carried out under conditions such that the cells have a doubling time of 160 minutes or less, and wherein the host cell culture has a volume of at least 10 L.

[0031] In another aspect of the present invention, a process for preparing a recombinant lectin is provided, the process comprising a) optionally, cloning the recombinant lectin gene into an expression vector and inserting the expression vector into a host cell; b) culturing the host cell in a suitable medium (wherein the culturing comprises a growth period carried out at a temperature of 25°C to 40°C and an expression period during which the recombinant lectin protein encoded by the recombinant lectin gene is expressed, wherein the expression period is carried out at a temperature of 15°C to 30°C and wherein the carbon to nitrogen ratio is maintained at 3:1 to 6:1 during the expression period); c) optionally, isolating the recombinant lectin protein expressed in (b) to obtain a crude recombinant lectin protein; and d) optionally, purifying the crude recombinant lectin protein to obtain an isolate of the recombinant lectin protein comprising.

[0032] In one embodiment, the expression vector in step a) is as shown in Figure 1. In another embodiment, the carbon source in step b is added to the culture medium at a rate of at least 0.5 g / L / h and no more than 2 g / L / h during the induction period, and the carbon source here is glucose or glycerol. Alternatively, the nitrogen source in step b is added to the culture medium at a rate of at least 0.4 g / L / h and no more than 1.5 g / L / h during the induction period, and the nitrogen source here is tryptone, peptone, or yeast extract.

[0033] In another embodiment, the isolation of the crude recombinant lectin protein in step "c" is performed by centrifugation followed by disruption of the cell surface. In a further embodiment, the purification of the crude recombinant lectin protein in step "d" includes at least one chromatographic step. In a further embodiment, at least one chromatographic step includes anion exchange chromatography and / or cation exchange chromatography. Alternatively, at least one chromatographic step includes hydrophobic interaction chromatography. In an additional embodiment, the purification of the crude recombinant lectin protein in step "d" includes a filtration step.

[0034] In some embodiments, the recombinant lectin protein is selected from an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% homology to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or any of these sequences. In some embodiments, the host cell culture medium has a volume of at least 10 L.

[0035] In yet another aspect of the present invention, a method for purifying crude recombinant lectin protein is provided, wherein the process is as follows: a) A step in which the crude recombinant lectin protein is first purified by anion exchange chromatography to obtain a first purified eluate; b) A step of optionally purifying the first purified eluate by hydrophobic interaction chromatography to obtain a second purified eluate; c) A step of optionally purifying the second purified eluate by cation exchange chromatography to obtain a third purified eluate; d) A step of further purifying the second or third purified eluate by anion exchange chromatography to obtain a fourth purified eluate; and e) Replacing the buffer of the fourth purified eluate by diafiltration to obtain a purified recombinant lectin protein isolate. Includes.

[0036] In some embodiments, the crude recombinant lectin protein is obtained by the method described above. In some embodiments, the recombinant lectin protein is selected from an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% homology to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or any of these sequences.

[0037] In some embodiments, the host cell culture medium has a volume of at least 10 L.

[0038] In a further aspect of the present invention, recombinant lectin proteins produced by the above method are provided.

[0039] Another aspect of the present invention provides a method for preparing recombinant lectin proteins having recombinant lectins with less than 20% initiation methionine, wherein the method is provided under the following conditions: a) Expression temperature of at least 15°C and 22°C or lower; b) The expression (i.e., induction) period lasts for at least 10 hours; c) The carbon source is added to the culture medium at a rate of 0.5–2 g / L / h; d) The nitrogen source is added to the culture medium at a rate of 0.4–1.5 g / L / h; e) A concentration of the derivative substance of at least 0.1 mM and 0.5 mM or less; f) Purifying the crude recombinant lectin protein using at least one chromatography step. It includes at least one of the following.

[0040] In a final aspect of the present invention, recombinant lectin proteins are provided, having recombinant lectins having less than 20% initiation methionine. That is, recombinant lectin protein mixtures are provided, wherein less than 20% of the recombinant lectin protein polymer has initiation methionine. In certain embodiments, the lectin protein polymer otherwise follows other aspects of the present invention. The present invention further provides recombinant lectin proteins prepared by the methods of the present invention. In particular, the present invention provides recombinant lectin proteins having recombinant lectins having less than 20% initiation methionine. [Brief explanation of the drawing]

[0041] [Figure 1] Annotated diagram of the pET27b vector having the nucleotide sequence of SEQ ID NO: 5, which codes for the amino acid sequence of SEQ ID NO: 1. [Figure 2] SDS-PAGE analysis of purified recombinant lectin having the amino acid sequence of SEQ ID NO: 1. [Figure 3] Western blot analysis of purified recombinant lectin having the amino acid sequence of SEQ ID NO: 1.

[0042] Table 1. Analysis of methionine lectin ratios obtained under various fermentation conditions. Table 2. Doubling time of E. coli under various fermentation conditions.

[0043] Detailed description of the invention As used herein, the term “doubling time” refers to the period of time required for the number of cells to double, for example, for one cell to increase to two cells, or for the number of cells in a population to double. As those skilled in the art will understand, the doubling time can be calculated by dividing the natural logarithm of 2 by the power exponent of the growth rate.

[0044] As used herein, the term “recombinant” refers to a genetically engineered product. Genetic engineering is understood to be the manipulation of genes that are not naturally occurring. Therefore, recombinant products are products that exist or are synthesized in a non-natural environment, such as a host cell, where the product does not naturally exist. As used herein, the term “protein” refers to a polymer of amino acid residues.

[0045] In the context of this invention, the term "growth period" will be understood to refer to the culture period during which host cells grow to a desired population density.

[0046] As used herein, the term “expression period” refers to the culture period during which recombinant lectins are expressed from host cells. The expression period can be distinguished from the proliferation period by the increased expression observed during the expression period compared to the proliferation period.

[0047] As used herein, the term "lectin" refers to a glycosylated protein.

[0048] As used herein, the term “amino acid” refers to natural amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimes that have functions similar to natural amino acids. Natural amino acids are those encoded by the genetic code, and these include proteogenic amino acids. Natural amino acids also include amino acids that have been post-translationally modified within cells. Synthetic amino acids include non-canonical amino acids, such as selenocysteine ​​and pyrrolidine. Typically, synthetic amino acids are not proteogenic amino acids.

[0049] As used herein, the terms “homology” or “homological” refer to two or more referenced entities that share at least partial identity over a given area or portion. An area, area, or domain of homology or identity refers to a portion of two or more referenced entities that share or are identical in homology or identity.

[0050] Therefore, if two sequences are identical across one or more sequence regions, they share identity in those regions. Substantial homology refers to molecules that are structurally or functionally conserved, and thus the molecule has or is expected to have at least one partial structure or function (e.g., biological function or activity) of the reference molecule, or of the relevant / corresponding region or part of the reference molecule with which the molecule shares homology.

[0051] According to a first aspect of the present invention, a method for preparing recombinant lectin proteins is provided, the method comprising the step of expressing recombinant lectin proteins encoded by recombinant lectin genes in a host cell culture medium, wherein the cell expression is carried out under conditions such that the cells have a doubling time of 160 minutes or less.

[0052] In some embodiments, the recombinant lectin gene is not a native gene. The recombinant lectin protein may not have the amino acid sequence of the native lectin protein. For example, the recombinant lectin protein may not contain or be derived from the amino acid sequence of SEQ ID NO: 2.

[0053] In some embodiments, cell expression is performed under conditions such that the cells have a doubling time of at least 100 minutes.

[0054] The inventors have discovered that higher levels of soluble protein can be obtained by expressing recombinant lectin proteins under culture conditions that limit the doubling time of host cells. Furthermore, it was found that such expression conditions also result in improved cleavage of initiating methionine from the recombinant lectin, thereby yielding a higher yield of protein lacking initiating methionine. By providing a process that results in the production of increased amounts of soluble protein with methionine already cleaved, this reduces the need for downstream processing of the protein, which would be necessary if a significant proportion of the protein were expressed as insoluble inclusions. This reduces process complexity, improves efficiency, and makes the process more economically viable.

[0055] As used herein, the term “soluble” refers to a modified lectin protein expressed in a soluble form or at least partially soluble form. In one embodiment, the solubility of a modified lectin protein is determined by cytolysis of a host cell expressing the modified lectin protein, followed by analysis of the lysis supernatant and pellet by SDS-PAGE. The presence of the modified lectin protein in the lysis supernatant indicates that it is soluble. The presence of the modified lectin protein in the lysis supernatant and pellet indicates that it is partially soluble. In one embodiment, as used herein, the term “soluble” refers to a modified lectin protein that does not form inclusion bodies. Using the above method, the presence of the modified lectin protein in the pellet indicates that it is expressed as an inclusion body.

[0056] As used herein, the term “cleavage of initiator methionine” refers to the removal of the N-terminal (initiator) methionine from an amino acid sequence. In one embodiment, cleavage of initiator methionine is catalyzed by the enzyme methionine aminopeptidase (MAP). In one embodiment, cleavage of initiator methionine is determined by mass spectrometry, which is known to those skilled in the art.

[0057] As used herein, the term “improved cleavage of initiating methionine” refers to an increase in the degree of cleavage of initiating methionine compared to a control. In one embodiment, it refers to an increase of at least 5%, 10%, 25%, or 50% in the degree of cleavage of initiating methionine compared to a control. In one embodiment, the control is the lectin protein of SEQ ID NO: 2. In some embodiments, recombinant lectin proteins (i.e., mixtures of lectin protein polymers) are provided, having recombinant lectins (i.e., met-lectins) having less than 50%, 40%, 30%, 20%, or 10% initiating methionine. Or, in other words, the methods of the present invention enable the production of recombinant lectin proteins (i.e., proteins lacking an initiating methionine residue) that are at least 50%, 60%, 70%, 80%, or 90% methionine-free.

[0058] It will be understood that the doubling time of host cells will be influenced by many factors, including culture temperature, culture medium, nutrient feed rate, and host cell type. Varying one or more of these parameters to achieve a desired doubling time will be within the capabilities of those skilled in the art.

[0059] One parameter that can be used to control the doubling time of host cells is the temperature at which expression takes place. In some embodiments, recombinant lectin expression is carried out at temperatures below 30°C, below 25°C, below 22°C, below 21°C, below 20°C, below 19°C, or below 18°C.

[0060] In some embodiments, recombinant lectin expression is carried out at a temperature of at least 15°C, at least 16°C, or at least 17°C (also referred to herein as the “expression temperature”). In some embodiments, the expression temperature is 18°C.

[0061] It has been found that expressing proteins at temperatures lower than 25°C, for example 18°C, is particularly advantageous because it helps to minimize the amount of protein produced, including the initiation methionine residue. This is especially true when the host cell is a mesophilic bacterium, such as E. coli. The fact that expression at lower temperatures, such as around 18°C, is beneficial is particularly surprising because it is much lower than the optimal growth temperature for host cells commonly used for recombinant protein expression, such as E. coli.

[0062] As is well known in the art, the temperature of a host cell culture medium can be controlled by placing the culture medium in a flask or bioreactor in an environment with a desired temperature, such as a water bath or a temperature-controlled room or chamber.

[0063] Before the expression of recombinant lectin proteins, host cells may be grown in a culture medium until they reach a desired population density. Therefore, in some embodiments, the method includes the step of culturing host cells, and the culturing is performed. -Proliferation period (during which the host cells are proliferated before protein expression) and - Includes the expression period (during which protein expression takes place).

[0064] During the proliferation phase, it may be desirable to induce rapid growth of host cells in order to maximize protein production during the subsequent expression phase. During the proliferation phase, the doubling time of host cells may be shorter than that of the expression phase (i.e., cells grow faster during the proliferation phase). For example, the doubling time of host cells during the proliferation phase may be less than 100 minutes. It will be understood that the doubling time of cells during the proliferation phase will vary depending on whether the cells are in a growth lag phase (during which the doubling time is relatively long) or an exponential growth phase (during which the doubling time is relatively short).

[0065] In some embodiments, the growth period is carried out at a higher temperature than the temperature at which the expression period takes place. In some embodiments, the growth period, or at least a portion thereof, is carried out at a temperature of at least 25°C, at least 30°C, or at least 35°C. In some embodiments, the growth period, or a portion thereof, is carried out at a temperature of 40°C or lower, or 38°C or lower.

[0066] Therefore, in some embodiments, the method will be understood to include a step of lowering the temperature of the culture medium from the growth period to the expression period. The temperature may be lowered gradually over a period of several hours. For example, the temperature may be lowered over a period of at least 2 hours, at least 4 hours, or at least 6 hours. In some embodiments, the temperature is lowered over a period of 6 or 7 hours or less.

[0067] The temperature may be lowered during the growth period. For example, the first growth period may be carried out at a first temperature (e.g., 30°C), and in the second growth period, the temperature may be lowered to the temperature for the expression period (e.g., from 30°C to 18°C).

[0068] The expression of recombinant lectin proteins can be initiated by the addition of an inducer to the culture medium. Therefore, the expression period can be defined as the stage during which the host cell culture medium contains the inducer. This period may also be called the "induction period."

[0069] As is well known in the art, inducers are molecules that regulate gene expression. Recombinant lectin genes may be under the control of an operator sequence. In the absence of an inducer, gene expression may be inhibited by the binding of a repressor to the operator sequence, or alternatively, by the lack of activation by an activator. In the presence of an inducer, repression of gene expression may be inhibited, or activation of gene expression may be possible. Therefore, by placing recombinant lectin genes under the control of an operator sequence regulated by an inducer, the expression of recombinant lectin proteins can be firmly controlled, thereby avoiding "skip" expression. Well-known examples of inducible expression systems include the ara operon and the lac operon. Binding of a repressor to the lac operator in the lac operon inhibits the transcription of downstream genes. Protein expression can be initiated using allolactose or its mimetic, IPTG (isopropyl β-D-1-thiogalactopyranoside), which binds to the lac repressor and releases it from the lac operator, thereby enabling transcription of the gene within the operon. Other inducible operons will be known to those skilled in the art.

[0070] Therefore, in some embodiments, the recombinant lectin gene is under the control of the lac operator. In such embodiments, the inducer comprises IPTG.

[0071] IPTG can be added to the culture medium at a concentration of at least 0.1 mM, at least 0.2 mM, or at least 0.25 mM.

[0072] In some embodiments, the concentration of IPTG is less than 1 mM. The concentration of IPTG may be 0.7 mM or less, or 0.5 mM or less. Using lower concentrations of IPTG may be beneficial to maintain low expression levels, which is thought to support efficient cleavage of initiation methionine.

[0073] Once the host cell population has increased to the desired density during the growth period, the inducer may be added. The host cell population can be determined by measuring the absorbance (OD) of the culture medium. In some embodiments, the inducer is added (i.e., the expression period is initiated) when the OD600 (absorbance of the culture medium measured at 600 nm) is at least 25, at least 30, at least 35, at least 38, or at least 40. In some embodiments, the inducer is added when the OD600 is at least 25 and 40 or less.

[0074] As is generally known in this art, batch cultured cells are first grown to obtain the desired cell aggregate. During this period, the focus is on amplifying cell proliferation rather than recombinant protein synthesis. Generally, the timing of induction is determined by the period when the desired cell aggregate is in active proliferation (during which time the cells have the maximum number of ribosomes for protein synthesis). After obtaining the desired cell aggregate, recombinant proteins will be produced after the addition of the inducer. Therefore, the timing of induction is crucial for the productivity of recombinant proteins. The timing of induction will vary depending on factors such as the type of host cell, the type of culture medium, the culture conditions, and the type of recombinant protein.

[0075] In some embodiments, the growth period is carried out for at least 4 hours, at least 6 hours, at least 8 hours, or at least 9 hours before the addition of the inducing substance that initiates the expression period.

[0076] The expression period (i.e., the culture time after the addition of the inducing substance) may be at least 10 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, or at least 35 hours. In some embodiments, the expression period is extended up to 40 hours.

[0077] When expression is performed at lower temperatures, a longer expression period is particularly beneficial because the cell proliferation rate is slower, and therefore the doubling time is longer. Consequently, the allowable expression period is extended to support more cell proliferation and compensate for the slower proliferation rate resulting from lower temperatures.

[0078] While I don't want to get bogged down in theory, it's thought that longer expression (induction) periods and extended doubling times allow for a steady increase in the amount of protein expressed. Depending on the metabolic conditions within the host cell, there may be sufficient time for methionine aminopeptidase (MAP) to cleave the initiating methionine, which helps maximize the yield of methionine-free recombinant protein.

[0079] Those skilled in the art will understand that they can select suitable culture conditions for a given host cell, including a suitable culture medium containing the nutrients necessary to support cell proliferation and protein expression. In some embodiments, cells are cultured in a liquid medium. Suitable liquid media for culturing microbial cells include lysogen / Luria broth (LB).

[0080] The carbon-to-nitrogen ratio in the culture medium is preferably maintained at a level between 3:1 and 6:1. This ratio can be maintained by controlling the feed rate of the culture medium.

[0081] The nutrient requirements necessary for cell proliferation and protein expression will be determined, at least in part, by factors such as cell population density, culture temperature, amount of inducer, and desired protein expression rate. Therefore, the rate at which nutrients are fed to cells can be used to help control the proliferation rate and, by extension, the protein expression rate.

[0082] In some embodiments, the carbon source is added to the culture medium at a rate of 2 g / L / h or less, or 1.5 g / L / h or less, during the expression period. The carbon source may also be added at a rate of at least 0.5 g / L / h, or at least 0.8 g / L / h, during expression.

[0083] The carbon source may include or consist of glycerol. Additionally or alternatively, other carbon sources such as glucose or dextrose may be used.

[0084] In some embodiments, the nitrogen source is added to the culture medium at a rate of 1.5 g / L / h or less, or 1 g / L / h or less, during the expression period. The nitrogen source may also be added at a rate of at least 0.4 g / L / h, or at least 0.6 g / L / h, during protein expression.

[0085] By restricting the supply of carbon and / or nitrogen to the parameters identified above, the rates of cell proliferation and protein expression were optimized, resulting in the expression of proteins in a substantially soluble form, with the majority of the proteins lacking initiation methionine. Conversely, uncontrolled feeding was found to result in increased protein expression rates and incomplete methionine cleavage.

[0086] In some embodiments, the method of the present invention enables the production of recombinant lectin proteins that are at least 80%, at least 85%, or at least 90% methionine-free (i.e., proteins lacking an initiating methionine residue).

[0087] In some embodiments, the method includes one or more or all of the following conditions: - An expression temperature of at least 15°C and no more than 22°C (e.g., 18°C); - The expression (i.e., induction) period takes at least 10 hours; - The carbon source is added to the culture medium at a rate of 0.5-2 g / L / h; - The nitrogen source is added to the culture medium at a rate of 0.4 to 1.5 g / L / h; - A concentration of the derivative substance of at least 0.1 mM and 0.5 mM or less.

[0088] A carbon source and / or a nitrogen source may be added to the culture medium during the expression period, during the growth period, or during both the expression and growth periods.

[0089] In some embodiments, the recombinant lectin protein is modified at at least one of the amino acids at positions 1, 14, 34, 113, and 123 of the natural SRL amino acid sequence (SEQ ID NO: 2), as described in International Publication No. 2010 / 095143, which is incorporated herein by reference. In some embodiments, the recombinant lectin protein is modified at all of these positions compared to the natural SRL.

[0090] Recombinant lectin proteins are (i) Sequence ID 1; (ii) Sequence ID 3; (iii) Sequence ID 4; or (iv) Amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NOs: 1, 3, or 4 It may contain or consist of an amino acid sequence selected from.

[0091] Sequence ID 1 represents a variant of the amino acid sequence of S. rolfsee's lectin (reported as Rec-2 in International Publication No. 2010 / 095143).

[0092] Sequence ID 2 shows the amino acid sequence of the lectin from natural S. rolfsee.

[0093] Sequence ID 3 represents a variant of the amino acid sequence of S. rolfsee's lectin (reported as Rec-3 in International Publication No. 2010 / 095143).

[0094] Sequence ID 4 shows a variant of the amino acid sequence of S. rolfsee's lectin (reported in International Publication No. 2014 / 203261).

[0095] In one embodiment, the "homology" ratio between two sequences is determined using the BLASTP algorithm with default parameters (Altschul et al. Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). In particular, the BLAST algorithm can be accessed on the internet using the URL: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. In an alternative embodiment, in global sequence alignment, the identity-homology ratio between two sequences is determined using the EMBOSS Needle algorithm with default parameters. In particular, the EMBOSS Needle algorithm can be accessed on the internet using the URL: https: / / www.ebi.ac.uk / Tools / psa / emboss_needle.

[0096] Unless otherwise specified, the term “homology” is used herein as synonymous with the term “identity.”

[0097] Recombinant lectin genes may contain or consist of the nucleotide sequence defined by Sequence ID No. 5. However, due to the degeneracy of the gene code, it will be understood that many alternative nucleotide sequences exist that can produce the same amino acid sequence.

[0098] In some embodiments, the recombinant lectin gene is contained within an expression construct, such as a plasmid or vector. The expression construct may further include a selection marker that allows selection of host cells having a construct encoding the recombinant lectin gene. The selection marker may be an antibiotic, such as kanamycin. In such embodiments, the culture medium may further include the antibiotic.

[0099] pET vectors are examples of artificial plasmids suitable for gene expression in bacterial cells, but other expression vectors will also be known to those skilled in the art. In some embodiments, the vector is pET27b.

[0100] In some embodiments, the host cell culture medium has a volume of at least 10 liters (L), at least 20 L, at least 30 L, at least 40 L, at least 50 L, or at least 100 L. Expression may be carried out in an industrial fermenter.

[0101] In some embodiments, the method further includes the step of cloning a recombinant lectin gene (e.g., a gene containing the sequence of Sequence ID No. 5) into an expression vector. Molecular cloning techniques are known to those skilled in the art and are described in texts such as "Molecular Cloning: A Manual of Experiments" by Sambroo and Russell.

[0102] The host cell may be a microbial cell, such as a bacterium, archaea, yeast, or fungus.

[0103] In some embodiments, the host cell is a bacterium, such as E. coli. A particularly preferred strain is E. coli BL21 DE3Gold.

[0104] In some embodiments, the method further includes the step of inserting an expression vector into host cells. This can be accomplished using methods commonly known to those skilled in the art, such as transformation or electroporation.

[0105] In some embodiments, the method further includes a step of isolating the expressed recombinant lectin protein. This isolation step yields a crude recombinant lectin protein (e.g., a protein containing the amino acid sequence of SEQ ID NO: 1). Isolation may include a centrifugation step, for example here, in which a cell pellet is obtained by centrifugation of the host cells. Isolation may further include resuspending the cell pellet in a buffer, suitable buffers for this purpose will be known to those skilled in the art. The isolation step may include lysis of the resuspended cell pellet by disrupting the cell membrane using a homogenizer, for example. Cell lysis may be carried out at a pressure of about 16,000 to 20,000 psi.

[0106] In some embodiments, the method further includes the purification of the crude recombinant lectin protein. Purification produces recombinant lectin protein isolates. Purification can be carried out by any suitable technique such as centrifugation (e.g., ultracentrifugation), size exclusion chromatography, ion exchange chromatography, electrophoresis, affinity chromatography, filtration (e.g., diafiltration), and high-performance liquid chromatography (HPLC) or a combination thereof.

[0107] Therefore, in some embodiments, the method is a) Depending on the case, a step of cloning a recombinant lectin gene into an expression vector and introducing the expression vector into host cells; b) Culturing host cells in a suitable medium (the cultivation comprising a growth period carried out at a temperature of 25°C to 40°C and an expression period during which recombinant lectin protein encoded by a recombinant lectin gene is expressed, the expression period carried out at a temperature of 15°C to 30°C, and the carbon-to-nitrogen ratio maintained at 3:1 to 6:1 during the expression period); c) If applicable, a step of isolating the recombinant lectin protein expressed in (b) to obtain a crude recombinant lectin protein; and d) Depending on the case, a step to purify the crude recombinant lectin protein to obtain an isolated recombinant lectin protein. Includes.

[0108] In some embodiments, the purification of the crude recombinant lectin protein includes at least one chromatographic step. In some embodiments, the at least one chromatographic step includes ion-effect chromatography and / or hydrophobic interaction chromatography.

[0109] In some embodiments, the purification of crude recombinant lectin protein is performed. i) A step in which the crude recombinant lectin protein is first purified by anion exchange chromatography to obtain a first purified eluate; ii) A step of optionally purifying the first purified eluate by hydrophobic interaction chromatography to obtain a second purified eluate; iii) A step of optionally purifying the second purified eluate by cation exchange chromatography to obtain a third purified eluate; iv) A step of further purifying the second or third purified eluate by anion exchange chromatography to obtain a fourth purified eluate; and v) Replacing the buffer of the fourth purified eluate by diafiltration to obtain a purified recombinant lectin protein isolate. Includes.

[0110] Advantageously, the above purification process allows recombinant lectin proteins to be obtained with high purity (97-99% pure) without the need for chemical or enzymatic treatment.

[0111] In a second aspect of the present invention, an expression construct comprising the nucleotide sequence of SEQ ID NO: 5 is provided.

[0112] A third aspect of the present invention provides a host cell comprising an expression construct of the second aspect of the present invention.

[0113] Unless otherwise specified, it will be understood that any embodiment described herein can be combined in any way with any aspect of the present invention.

[0114] In some specific embodiments, the present invention relates to a process for preparing recombinant lectin proteins, such as a protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the process is a. A step of preparing clones by cloning the nucleotide sequence encoding the recombinant lectin protein (the sequence of SEQ ID NO: 5) in recombinant host cells while it is embedded in the vector. b. A step of obtaining a fermentation product by fed-batch fermentation of clones prepared in step "a" for the expression of recombinant lectin protein (e.g., having the amino acid sequence of SEQ ID NO: 1) in a suitable medium, with an initial temperature of approximately 25°C to approximately 40°C and an induction period temperature of approximately 15°C to 30°C, and a feed rate such that the carbon source to nitrogen source ratio is 3:1 to 6:1; c. Isolation and clarification of the fermentation product obtained in step "b" to obtain a crude recombinant lectin protein (for example, having the amino acid sequence of SEQ ID NO: 1); and d. Purification of the crude recombinant lectin protein obtained in step "c" (for example, having the amino acid sequence of SEQ ID NO: 1). It includes. In some cases, purification is performed. i. A step of first purifying a crude recombinant lectin protein (for example, having the amino acid sequence of SEQ ID NO: 1) by anion exchange chromatography to obtain a first purified eluate; ii. A step in which the first purified eluate obtained from step i is optionally purified by hydrophobic interaction chromatography to obtain a second purified eluate; iii. A step of purifying the second purified eluate obtained from step ii or the first purified eluate obtained from step i by cation exchange chromatography to obtain a third purified eluate; iv. A step of further purifying the third purified eluate obtained from step iii or the second purified eluate obtained from step ii by anion exchange chromatography to obtain a purified eluate; v. The process includes replacing the buffer of the purified eluate obtained from step iv by diafiltration to obtain a recombinant lectin protein (for example, having the amino acid sequence of SEQ ID NO: 1) as a pure product.

[0115] The present invention further relates to fed-batch fermentation of recombinant bacterial cells in a suitable culture medium, wherein the feed rate of the carbon source during fermentation to obtain the fermentation product is 0.5 to 2 g / L / h, and the feed rate of the nitrogen source is 0.4 to 1.5 g / L / h.

[0116] The present invention further relates to a purification process for crude recombinant lectins, such as proteins having the amino acid sequence of SEQ ID NO: 1, wherein the process is: a. A step of first purifying a crude recombinant lectin protein (for example, having the amino acid sequence of SEQ ID NO: 1) by anion exchange chromatography to obtain a first purified eluate; b. A step in which the first purified eluate obtained from step a is optionally purified by hydrophobic interaction chromatography to obtain a second purified eluate; c. A step of purifying the second purified eluate obtained from step b or the first purified eluate obtained from step a by cation exchange chromatography to obtain a third purified eluate; d. A step of further purifying the third purified eluate obtained in step c or the second purified eluate obtained in step b by anion exchange chromatography to obtain a purified eluate; e. The process includes replacing the buffer of the purified eluate obtained from step d by diafiltration to obtain a recombinant lectin protein (for example, having the amino acid sequence of SEQ ID NO: 1) as a pure product.

[0117] The present invention also relates to clones used for the production of recombinant lectins having the amino acid sequence of SEQ ID NO: 1, which, while embedded in a vector, contain the nucleotide sequence of SEQ ID NO: 5 encoding the amino acid sequence of SEQ ID NO: 1 in recombinant host cells.

[0118] The present invention further relates to a recombinant lectin having the amino acid sequence of SEQ ID NO: 1, as prepared by the process disclosed herein.

[0119] Advantageously, in some embodiments, the process of the present invention yields the desired product in a yield of over 90%, over 95%, or over 97%.

[0120] The purity of recombinant lectin protein products may be at least 90%, at least 95%, at least 97%, or at least 99% when measured using standard methods such as anion exchange chromatography.

[0121] The present invention relates to a process for preparing a recombinant lectin having the amino acid sequence of SEQ ID NO: 1, wherein the process is: a. A step of preparing a clone by cloning the nucleotide sequence of sequence number 5, which codes for the amino acid sequence of sequence number 1, in recombinant host cells while it is embedded in the vector. b. A step of obtaining a fermentation product by fed-batch fermentation of clones prepared in step "a" for the expression of recombinant lectin having the amino acid sequence of SEQ ID NO: 1, in a suitable medium, with an initial temperature of approximately 25°C to approximately 40°C and an induction period temperature of approximately 15°C to 30°C, and a feed rate such that the carbon source to nitrogen source ratio is 3:1 to 6:1; c. Isolation and clarification of the fermentation product obtained in step "b" to obtain a crude recombinant lectin having the amino acid sequence of SEQ ID NO: 1; and d. Purification of the crude recombinant lectin having the amino acid sequence of SEQ ID NO: 1 obtained in step "c" (This purification is performed by i. A step in which a crude recombinant lectin having the amino acid sequence of SEQ ID NO: 1 is first purified by anion exchange chromatography to obtain a first purified eluate; ii. A step in which the first purified eluate obtained from step i is optionally purified by hydrophobic interaction chromatography to obtain a second purified eluate; iii. A step of purifying the second purified eluate obtained from step ii or the first purified eluate obtained from step i by cation exchange chromatography to obtain a third purified eluate; iv. A step of further purifying the third purified eluate obtained in step iii or the second purified eluate obtained in step ii by anion exchange chromatography to obtain a purified eluate; (v. A step comprising replacing the buffer of the purified eluate obtained from step iv by diafiltration to obtain recombinant lectin having the amino acid sequence of SEQ ID NO: 1 as a pure product.) Includes.

[0122] Cloning and expression of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 According to aspects of the present invention, the host cell used for the production of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 may be a bacterial cell or a yeast. A preferred bacterial cell is E. coli, particularly the E. coli BL21 DE3 Gold strain. The nucleotide sequence of SEQ ID NO: 5 encoding the recombinant lectin having the amino acid sequence of SEQ ID NO: 5 can be cloned into a vector, preferably pET27b. The plasmid can then be transformed and expressed in the host E. coli BL21 DE3 Gold.

[0123] Fermentation process In an exemplary embodiment, the E. coli BL21 DE3 Gold strain, containing plasmid pET27b containing a nucleotide sequence encoding a recombinant lectin protein, is grown in an inoculum containing Luria HiVeg broth (20 g / l), Na2HPO4 (7.5 g / l), dextrose (5 g / l), MgSO4·7H2O (1 g / l), kanamycin to a final concentration of 20 μg / ml, and a 0.1% (v / v) trace metal solution in hydrochloric acid of FeSO4, ZnSO4, CoCl2, NaMoO4, CaCl2, MnCl2, CuSO4, or H3BO3. The inoculum is prepared by growing cells at 30-40°C for 12-16 hours. Fed-batch fermentation of recombinant E. coli BL21 DE3 Gold strain may be carried out in a production medium containing yeast extract (10 g / l), dextrose (12 g / l), KH2PO4 (3 g / l), K2HPO4 (12.5 g / l), (NH4)2SO4 (5 g / l), NaCl (0.5 g / l), MgSO4·7H2O (1 g / l), and 0.1% (v / v) trace metal solutions in hydrochloric acid of FeSO4, ZnSO4, CoCl2, NaMoO4, CaCl2, MnCl2, CuSO4, or H3BO3. Kanamycin may be added until a final concentration of 20 μg / ml is reached. Feeding may be initiated using a suitable carbon source, such as glucose or glycerol, preferably 50% (w / v) glycerol, and a nitrogen source, such as tryptone, peptone, or yeast extract, preferably 40% (w / v) yeast extract. Feeding may be initiated after log 5 hours at a predetermined feed rate. While maintaining a C:N ratio in the range of 3:1 to 6:1, preferably 4:1, the feed rate of the carbon source during fermentation may be 0.5 to 2 g / L / h, and the feed rate of the nitrogen source may be 0.4 to 1.5 g / L / h. Those skilled in the art may change the ratios and amounts according to their suitability, for they are specific to the parameters shown for a particular batch size.

[0124] The initial growth period may be carried out at 25°C to 40°C, and the induction period at 15°C to 30°C, followed by a series of purifications by column chromatography. The temperature may be maintained at approximately 25°C to 40°C, preferably at approximately 37°C, during the initial growth period. The temperature may be lowered during the induction period and may be maintained at approximately 15°C to 30°C, preferably at approximately 22°C.

[0125] Initial growth in the culture medium can be carried out using an aeration rate of 1-2 vvm, dissolved oxygen maintained at 50-60%, and a pH maintained at 6.6-7.2 with an alkali such as sodium hydroxide. The total culture time may be 20-50 hours, or 30-40 hours, preferably about 36 hours. Feeding of carbon and nitrogen sources may be continued until the end of the culture run.

[0126] Expression of recombinant lectin proteins (e.g., having the amino acid sequence of SEQ ID NO: 1) can be performed by inducing a host cell (e.g., E. coli) culture broth with an inducer such as lactose or isopropylthio-galactopyranoside (IPTG), preferably at a concentration of about 50 μM to 1500 μM, preferably about 1000 μM. The culture can be induced at a cell density measured at least about 20-50, preferably about 30-50, at about 600 nm.

[0127] Recombinant lectin protein expression can be demonstrated by SDS-PAGE analysis of the whole cell lysate and lysate supernatant. Recombinant protein expression may be present at approximately 50% to 60% of the total protein and may be expressed in the cytoplasm in a soluble form. The yield of recombinant lectin protein, as analyzed by SDS-PAGE, can be at least approximately 5–9 g / L (fermentation broth).

[0128] Isolation of soluble recombinant lectin proteins can be performed by collecting cells by centrifugation to obtain a cell pellet, and then resuspending the cells in a suitable buffer, which may be pre-cooled to a temperature of approximately 6°C to 10°C. Cell disruption may be performed in some cases under high pressure of approximately 16,000 to 20,000 psi using a homogenizer (MiniDebee). The resulting cell lysates can then be further processed for purification of soluble recombinant lectin proteins, for example, by using various chromatographic processes.

[0129] Purification of recombinant lectin proteins The purification process for recombinant lectin proteins obtained from cell lysates resulting from the fermentation process may include the following steps: a Column 1: Anion exchange chromatography, b Column 2: Hydrophobic interaction chromatography, c Column 3: Cation exchange chromatography, d Column 4: Anion exchange chromatography.

[0130] In some embodiments, a clear solution is obtained by clarifying the total cell lysate obtained after cell lysis using a tangential flow filtration system of approximately 0.1 μm.

[0131] In some embodiments, the clarified cell lysate is subjected to anion exchange column chromatography using resins such as Cellfine Max Qr, Source 30Q, Source 15Q, and DEAE Sepharose. The column may be equilibrated with a suitable buffer, for example, a buffer having about 10–30 mM Tris, about 0.5–2 mM EDTA, and a pH of about 7.5–9.0. After loading, the column may be washed with a buffer having a conductivity in the range of about 4–8 mS / cm. Elution may be performed with a buffer containing about 10–30 mM Tris, about 0.5–2 mM EDTA, and sodium chloride having a conductivity of about 15–20 mS / cm.

[0132] In some embodiments, the proteins obtained from the anion exchange column may be subjected to hydrophobic interaction column chromatography. The resins used may include Cellfine Max butyl, butyl Sepharose, and phenyl Sepharose. The column may be equilibrated with approximately 20-30 mM sodium acetate buffer containing approximately 0.5-2 M ammonium sulfate and approximately 0.5-2 mM EDTA, with a pH of approximately 4-5. Elution may be performed with approximately 20-30 mM sodium acetate buffer containing approximately 0.5-2 mM EDTA and approximately 10-20 g / l ammonium sulfate, with a pH in the range of approximately 4-5 and a conductivity in the range of approximately 2-100 ms / cm. After hydrophobic interaction column chromatography or the eluate obtained from the anion exchange column may then be subjected to cation exchange column chromatography using resins such as SP Sepharose, CM Sepharose, and Cellfine Max Sh. The column can be equilibrated with approximately 20-30 mM sodium acetate buffer containing approximately 0.5-2.0 mM EDTA and having a pH in the range of approximately 4-5. The elution buffer may contain approximately 20-30 mM sodium acetate, approximately 0.5-2 mM EDTA, and approximately 0.3-1 M sodium chloride, with a pH in the range of approximately 4-5. A stepwise gradient in the range of approximately 15-25% may be applied before elution. Elution can be performed using a stepwise gradient in the range of approximately 50-80%.

[0133] In some embodiments, the eluate obtained by cation exchange chromatography is diluted with water for injection (WFI) or purified water (PW) in a ratio of approximately 1:2 to 1:5. The pH of the protein solution can then be adjusted to approximately 7.5–8.5 using sodium hydroxide, Tris buffer, or glycine buffer (pH 11.0), and subsequently the buffer is replaced with Tris buffer (pH 7.5–8.5) using a tangential flow filtration system until the conductivity is reduced to within the range of approximately 1–5 mS / cm. The protein solution can then be subjected to anion exchange column chromatography. Resins such as Cellfine Max Qr, Source Q15, Source Q30, or DEAE Sepharose can be used. The column can be equilibrated with approximately 20–30 mM Tris buffer having a pH in the range of approximately 7.5–8.5. Elution may be performed using a buffer solution containing approximately 20–30 mM Tris and approximately 0.3–1 M sodium chloride, with a pH in the range of approximately 7.5–8.5. A linear gradient of up to approximately 15% of a column volume of 3–20 may be applied for elution. The eluate thus obtained can be exchanged for a suitable buffer solution, such as Tris-buffered saline, phosphate-buffered saline, or acetate buffer, by dialysis filtration using a 3, 5, or 10 kDa cutoff membrane.

[0134] The biological activity of the protein can be maintained by storing the buffer-exchanged protein at approximately 2-8°C. Alternatively, the eluate obtained after the second anion exchange chromatography can be buffer-exchanged with water for injection, and then freeze-dried to obtain a powdered protein.

[0135] The purity of recombinant lectin proteins can be confirmed by anion-exchange high-performance liquid chromatography. Recombinant lectin proteins with the amino acid sequence of SEQ ID NO: 1, obtained after a series of chromatographic separations, were found to be 97% to 99% pure. The proportion of recombinant lectin proteins with the amino acid sequence of SEQ ID NO: 1 that contained unprocessed starter methionine was found to be approximately 10–15% (compared to the amount of methionine-free lectin).

[0136] Therefore, the present invention provides a method for expressing recombinant lectin proteins, such as SEQ ID NO: 1, in a vector that provides robust regulation of protein expression and soluble expression in host cells. The process of the present invention is controllable and scalable, overcoming the shortcomings of previous processes. Therefore, the present invention describes an industrially scalable process for producing gram quantities of recombinant lectin.

[0137] Therefore, the main objective of the present invention, namely, overcoming the shortcomings of prior art objects and devising a highly efficient process for preparing the amino acid sequence of SEQ ID NO: 1, is achieved.

[0138] The yield of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 can be 1.5 to 3.0 g / L (fermentation broth). Therefore, another objective of the present invention, namely providing a process that yields very high yields, is cost-effective, and can be carried out using readily available raw materials, is also achieved. The process of the present invention is cost-effective because the raw materials required to prepare a large amount of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 are quite small. Furthermore, the process does not require very high or low temperatures or expensive equipment to carry out. Therefore, the process is cost-effective and can be easily scaled up.

[0139] The present invention further relates to fed-batch fermentation of recombinant bacterial cells in a suitable culture medium, wherein the feed rate of the carbon source during fermentation to obtain the fermentation product is approximately 0.5 to 2 g / L / h, and the feed rate of the nitrogen source is approximately 0.4 to 1.5 g / L / h.

[0140] The initial fermentation / growth period can be carried out at approximately 25°C to 40°C, after which the temperature is lowered to approximately 15°C to 30°C during the induction period, followed by a series of purifications by column chromatography.

[0141] In some embodiments, initial growth of the culture medium may be carried out using an aeration rate of about 1–2 vvm, dissolved oxygen maintained at about 50–60%, and pH maintained at about 6.6–7.2 with an alkali such as sodium hydroxide. In some embodiments, the temperature maintained during initial growth was about 25°C–40°C. The temperature during the induction period may be lowered or maintained at about 15°C–30°C. The fermentation run time may be at least about 20–50 hours. The feeding of carbon and nitrogen sources may be continued until the end of the fermentation run time.

[0142] In some embodiments, the clarified cell lysate is subjected to anion exchange column chromatography using resins such as Cellfine Max Qr, Source 30Q, Source 15Q, and DEAE Sepharose. The column may be equilibrated with approximately 10–30 mM Tris buffer, approximately 0.5–2 mM EDTA, and a buffer having a pH of approximately 7.5–9.0. After loading, the column may be washed with a buffer having a conductivity in the range of approximately 4–8 mS / cm. Elution may be performed with a buffer containing 10–30 mM Tris, approximately 0.5–2 mM EDTA, and sodium chloride having a conductivity of approximately 15–20 mS / cm.

[0143] In some embodiments, the protein obtained from the anion exchange column may be subjected to hydrophobic interaction column chromatography. The resins used may include Cellfine Max butyl, butyl Sepharose, and phenyl Sepharose. The column may be equilibrated with 20-30 mM sodium acetate buffer containing approximately 0.5-2 M ammonium sulfate and approximately 0.5-2 mM EDTA, with a pH of approximately 4.0-5.0. Elution may be performed using approximately 20-30 mM sodium acetate buffer having approximately 0.5-2 mM EDTA, approximately 10-20 g / L ammonium sulfate, a pH in the range of approximately 4-5, and a conductivity in the range of approximately 2-100 ms / cm. The eluate obtained after hydrophobic interaction column chromatography or from the anion exchange column may then be subjected to cation exchange column chromatography using resins such as SP Sepharose, CM Sepharose, and Cellfine Max Sh. The column can be equilibrated with approximately 20-30 mM sodium acetate buffer containing approximately 0.5-2.0 mM EDTA and having a pH in the range of approximately 4-5. The elution buffer may contain approximately 20-30 mM sodium acetate, approximately 0.5-2 mM EDTA, and approximately 0.3-1 M sodium chloride, and may have a pH in the range of approximately 4-5. A stepped gradient in the range of approximately 15-25% may be applied before elution. Elution may be performed using a stepped gradient in the range of approximately 50-80%.

[0144] The eluate obtained by cation exchange chromatography can be diluted to a ratio of approximately 1:2 to 1:5 using sterile water for injection or purified water. The pH of the protein solution can then be adjusted to approximately 8 using NaOH, Tris buffer, or glycine buffer (pH 11.0). Subsequently, buffer exchange using Tris buffer (pH 7.5-8.5) with a tangential flow filtration system may be continued until the conductivity is reduced to within the range of approximately 1-5 mS / cm. The protein solution can then be subjected to anion exchange column chromatography. Resins such as Cellfine Max Qr, Source Q15, Source Q30, or DEAE Sepharose may be used. The column can be equilibrated with approximately 20-30 mM Tris buffer having a pH in the range of approximately 7.5-8.5. Elution can be performed using a buffer containing approximately 20-30 mM Tris and approximately 0.3-1 M sodium chloride, with a pH in the range of approximately 7.5-8.5. For elution, a linear gradient of up to approximately 15% can be applied with a column volume of 3 to 20. The eluate thus obtained can be re-buffered with a suitable buffer such as Tris-buffered saline, phosphate-buffered saline, or acetate buffer by diafiltration using a 3, 5, or 10 kDa cutoff membrane. The re-buffered protein can maintain its biological activity by being stored at 2 to 8°C. Alternatively, the eluate obtained after a second anion exchange chromatography can be re-buffered with water for injection, and then lyophilized to obtain the protein in powder form. The purity of recombinant lectins having the amino acid sequence of SEQ ID NO: 1 can be confirmed by anion exchange high-performance liquid chromatography. Recombinant lectins having the amino acid sequence of SEQ ID NO: 1 obtained in this way after separation by chromatography of this series are 97% to 99% pure. The proportion of recombinant lectins having the amino acid sequence of SEQ ID NO: 1 with unprocessed initiating methionine was 10 to 15% (compared to the amount for methionine-free lectin).

[0145] The present invention relates to a clone used for the production of a recombinant lectin having the amino acid sequence of SEQ ID NO: 1, which includes the nucleotide sequence of SEQ ID NO: 5, encoding the amino acid sequence of SEQ ID NO: 1, in a recombinant host cell while embedded in a vector.

[0146] Examples The examples are provided to demonstrate the best mode of carrying out the present invention and do not in any way limit the scope of the invention.

[0147] Example 1: Cloning of lectin SEQ ID NO: 5 in pET27b vector and expression in E. coli BL21 DE3 A nucleotide sequence (SEQ ID NO: 5) encoding a recombinant lectin having the amino acid sequence of SEQ ID NO: 1, pre-cloned into a pET20b vector (as disclosed in patent no. WO2010 / 095143A2), was subcloned into a pET27b vector. E. coli BL21 DE3 cells containing SEQ ID NO: 5 cloned into pET20b were grown in Luria Hiveg broth (Himedia, Mumbai, India). Plasmids were isolated from cells using the GeneJET Plasmid MiniPrep Kit (Thermo Scientific) according to the manufacturer's instructions. Plasmid pET20b was digested with restriction enzymes NdeI and BamHI (New England Biolabs). The digested plasmids were run on an agarose gel electrophoresis, and the separated insertion fragments were eluted from the gel using the GeneJET Gel Extraction Kit (Thermo Scientific). A similar vector, pET27b, was isolated and digested with the same restriction enzymes (NdeI and BamHI). The insertion fragment (SEQ ID NO: 5) isolated from pET20b was cloned into the pET27b vector (pET27b-Lec) and transformed into E. coli BL21 DE3 (Gold). Clones were screened by colony PCR, and positive clones were further used for expression analysis. Positive clones were grown in Hiveg Luria broth at 37°C, induced with 0.25 mM IPTG at a cell density of 1–1.2 (OD600 nm), and further grown for 4 hours. Expression of recombinant lectin with the amino acid sequence of SEQ ID NO: 1 was confirmed by SDS-PAGE analysis. The sequence of the insertion fragment in vector pET27b was confirmed by DNA sequencing of the isolated plasmid. Glycerol stocks of positive clones were prepared and maintained at -80°C.

[0148] Example 2: Fermentation Process The culture medium from the glycerol stock was inoculated into a culture medium containing 2% Hiveg Luria broth, 0.75% Na2HPO4, 0.5% dextrose, and kanamycin (20 μg / ml). The culture medium was grown at 30 ± 2°C and 110 rpm for 16 hours. Approximately 300 ml of the culture medium was inoculated into 2.3 L of a production medium containing 1% yeast extract (w / v), 1.2% dextrose (w / v), 0.3% KH2PO4 (w / v), 1.25% K2HPO4 (w / v), 0.5% (NH4)2SO4 (w / v), 0.05% NaCl (w / v), 0.1% MgSO4·7H2O (w / v), 0.1% (v / v) trace metal solution, and kanamycin (20 μg / ml). Fermentation was carried out in a 5L fermenter (Biostat B, Sartorium Stedim) with an aeration rate of 1-2 vvm, maintaining dissolved oxygen at 50-60% and pH at 6.6-7.0 using alkali. Initial growth was carried out at a temperature of 37°C. The temperature was gradually reduced and maintained at 22°C during the induction period. The carbon source (glycerol) and nitrogen source (yeast extract) were fed at a predetermined feed rate after 5 log hours, while maintaining a C:N ratio within the range of 4:1. The culture medium was approximately 45 (OD 600 Fermentation was induced using 1 mM IPTG at a cell density of ). Fermentation was continued for up to 24 hours, and the culture broth was collected by centrifugation at 9000 rpm for 15 minutes. The wet weight of the cell aggregate obtained from the fermentation broth was 372 g.

[0149] Example 3: Isolation of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 from cells The cell pellet was suspended in lysis buffer (25 mM Tris, 1 mM EDTA, pH 8.5) in a 1:10 (w / v) ratio and stirred with an overhead stirrer for at least 2 hours to form a homogeneous suspension. The suspension was lysed by high-pressure homogenization at approximately 18,000 psi. The cell lysate was centrifuged at 9,000 rpm for 15 minutes at 15°C. The resulting supernatant was retained and further processed for purification of recombinant lectin having the amino acid sequence of SEQ ID NO: 1.

[0150] Example 4: Removal of nucleic acid impurities and clarification of protein solution by microfiltration The supernatant was treated with at least 0.025% polyethyleneimine by stirring for 15–30 minutes. The total cell lysate obtained after polyethyleneimine treatment was pre-equalized with 25 mM Tris buffer containing 1 mM EDTA and pH 8.0 ± 0.5 at 3600 cm³. 2 The solution was clarified using a 0.1 μm hollow fiber. An intermembrane pressure differential of 5–10 psi was maintained throughout the clarification process. The residue was dialyzed using the equilibrium buffer described above in a stepwise mode to recover recombinant lectin having the amino acid sequence of SEQ ID NO: 1 from the filtration permeate. A recovery rate of over 90% of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 was obtained in the filtration permeate. 84.4 g of total protein was recovered in the filtration permeate, as measured by absorbance at 280 nm. The purity of the protein of interest was measured by HPLC and was 49.4%.

[0151] Example 5: Purification of protein by ion exchange chromatography (column 1) The clarified protein solution was loaded onto a Cellfine Max Qr resin that had been pre-equilibrated with 25 mM Tris buffer containing 1 mM EDTA and a pH of 8.0 ± 0.5. After loading, the column was washed with equilibrium buffer, followed by washing with equilibrium buffer containing 1–3 g / L of NaCl. The protein was eluted using 25 mM Tris buffer containing 1 mM EDTA and 11–15 g / L of NaCl and a pH of 8 ± 0.5. The total protein recovered from this column was 41.6 g, with a purity of 77.4% and was active protein.

[0152] Example 6: Protein precipitation and purification by hydrophobic interaction chromatography (HIC-column 2) The pH of the eluate from column 1 was adjusted to 4.5 using acetic acid, and then precipitated with ammonium sulfate. The solution was then centrifuged, and the clear supernatant was treated for further purification on hydrophobic interaction chromatography resin (Cellfine Max butyl). The column was equilibrated with 25 mM sodium acetate containing 1 mM EDTA and 0.5–2 M ammonium sulfate at pH 4.5, and then the protein solution was loaded. After loading, washing with equilibrium buffer was performed, followed by elution using elution buffer containing 25 mM sodium acetate, 1 mM EDTA, and 15 g / L ammonium sulfate at pH 4.5. 27.5 g of total protein with a purity of 90.3% was eluted from column 2.

[0153] Example 7: Purification of proteins by cation exchange chromatography (column 3) The protein obtained from column 2 was diluted with purified water to a conductivity of approximately 20 mS / cm and loaded onto SP Sepharose FF resin pre-equilibrated with 25 mM sodium acetate buffer containing 1 mM EDTA and pH 4.5. After loading, the column was washed with equilibrium buffer, and then eluted with a 20% stepped gradient elution buffer containing 25 mM sodium acetate, 1 mM EDTA, and 0.5 M NaCl and pH 4.5. Elution was performed by passing a stepped gradient of elution buffer from 50 to 70%. A total of 24.6 g of protein with a purity of 93.0% was recovered from the column. The elution buffer of column 3 was changed on a 3 kDa membrane, and the protein was obtained in 25 mM Tris buffer (pH 8.0).

[0154] Example 8: Purification of proteins by anion exchange chromatography After changing the buffer, 20.9 g of protein was obtained and processed on Source 30Q resin. The column was equilibrated with 25 mM Tris buffer (pH 8.0), and then the protein solution was loaded. After loading, the column was washed with equilibration buffer. Elution was then performed by flowing a linear gradient elution buffer containing 25 mM Tris and 0.5 M NaCl, at pH 8.0, in a column volume of 15. One peak was obtained, which showed a purity of over 99.0% when analyzed by HPLC. Assuming that 1 OD at 280 nm is equivalent to 1 mg, the total amount obtained was measured by absorbance at 280 nm, yielding 9.0 g. The final eluate was buffer-changed with TBS buffer (50 mM Tris buffer, 150 mM NaCl, pH 7.8).

[0155] Example 9: Physicochemical characteristics of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 The purity of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 was determined by SDS-PAGE and HPLC analysis. HPLC analysis showed a purity of 97–99% for recombinant lectin having the amino acid sequence of SEQ ID NO: 1. SDS-PAGE analysis of recombinant lectin having the amino acid sequence of SEQ ID NO: 1 showed a single band with a molecular weight of approximately 16 kDa. Protein identity was confirmed by Western blotting, and biological activity was confirmed by hemagglutination assays and in vitro cell-based assays using various cancer cell lines. The molecular weight of the purified recombinant lectin having the amino acid sequence of SEQ ID NO: 1 was 16044 daltons.

[0156] Example 10: Fermentation process conditions The glycerol stock culture solution described in previous examples was inoculated into a medium containing 2% Hiveg Luria broth, 0.75% Na2HPO4, 0.5% dextrose, and kanamycin (20 μg / ml). The culture solutions from batches 1-3 were grown at 37°C and 110 rpm for 16 hours.

[0157] For batches 4 and 5, the glycerol stock culture solution described in the previous example was inoculated into the culture medium as described above, except that the culture solution was grown at 30°C and 110 rpm for 19 hours.

[0158] Subsequently, approximately 300 ml of culture medium was inoculated into 2.3 L of production medium.

[0159] The production culture medium was the same as described in Example 2 (and as shown in Table 1), except for the following differences between batches.

[0160] [Table 1]

[0161] Batches 1-3: The production medium contained 12 g / L of dextrose, according to Example 2.

[0162] Batches 4-5: The production medium contained 10 g / L of dextrose instead of the 1.2% dextrose described in Example 2.

[0163] For each batch, fermentation was carried out in a 5L fermenter (Biostat B, Sartorium Stedim) with an aeration rate of 1-2 vvm, with dissolved oxygen maintained at 50-60%, and pH maintained at 6.6-7.0 using alkali.

[0164] The temperature of the production medium was initially maintained at 37°C using the following conditions.

[0165] For batches 1 and 2, the temperature was gradually lowered to 22°C every log4 hours. This temperature was maintained during the induction period, which was initiated using 1 mM IPTG. The total batch execution time was 25 hours.

[0166] The feeding of the carbon source (glycerol) and nitrogen source (yeast extract) was started after 4 log hours. The total amount of glycerol fed to batch 1 was 102 g / L, and 75 g / L was fed to batch 2.

[0167] Batch 3 – The temperature was gradually lowered to 18°C ​​by the 5th hour. This temperature was maintained during the induction period, which was initiated using 1 mM IPTG. The total batch execution time was 25 hours.

[0168] The total amount of glycerol fed into batch 3 during the induction period was 70 g / L.

[0169] In batches 1-3, the inducer was added to the culture medium 4-6 hours after inoculation. The addition of the inducer initiated the induction (expression) period.

[0170] Batch 4-5 - After inoculation into the production medium, the temperature was gradually lowered to 18°C ​​by the 6th hour. This temperature was maintained during the induction (expression) period, which was initiated 9 hours after inoculation using 0.25 mM IPTG. The total batch run time was either 33 hours (batch 8) or 48 hours (batch 9).

[0171] The total amount of glycerol fed into batch 4 during the induction period was 35 g / L, while the total amount of glycerol fed into batch 5 during the induction period was 40 g / L.

[0172] The culture broth was collected by centrifugation at 9000 pm for 15 minutes, and recombinant lectins were isolated and purified according to the present invention. The ratio of methionine lectins in the isolated lectins was then analyzed.

[0173] Over 50% of the lectins isolated in batch 1 were methionine-lectins. This decreased to 26% in batch 2, 16% in batch 3, 13% in batch 4, and 12% in batch 5.

[0174] Example 11: Doubling time The doubling time of E. coli was determined at various time points and temperatures during the sample fermentation process, i.e., after inoculation into the production medium. The results are shown in Table 2. In this example, the inducer, IPTG in this embodiment, was added 9 hours after inoculation. Therefore, in this example, the growth period will be understood to be before 9 hours, and the expression period will be understood to be at the time of and after the addition of the inducer.

[0175] [Table 2]

[0176] Example 12: Purification of recombinant lectin In this example, recombinant lectins are isolated and purified from culture broth, such as that produced in Example 10.

[0177] The culture broth is centrifuged at 9000 rpm for 15 minutes at 15°C. The resulting pellet is resuspended in lysis buffer (25 mM Tris, 1 mM EDTA, pH 8.0). The cells are lysed by high-pressure homogenization at 18000 psi. The lysate is clarified using 0.1 μm hollow fibers pre-equilibriumized with lysis buffer. The clarified protein solution is subjected to a series of chromatographic steps to purify recombinant lectins.

[0178] Anion exchange chromatography: The clarified protein solution is loaded onto a Cellfine Max Qr column equilibrated with 25 mM Tris, 1 mM EDTA, and pH 8.0 at a binding strength of 60–80 mg / ml. After loading, the column is washed with 2–3 column volumes of equilibrium buffer. The column is then washed again with equilibrium buffer containing sodium chloride with a conductivity of approximately 5 mS / cm. Elution of the bound protein is performed using 25 mM Tris buffer, 1 mM EDTA, and pH 8.0, which contains sodium chloride with a conductivity of 18–20 mS / cm. All peaks are recovered as a single fraction, which contains the protein of interest along with some other impurities.

[0179] Cation exchange chromatography: The eluate from anion exchange chromatography is subjected to cation exchange chromatography using an SP Sepharose FF column. The pH of the eluate is adjusted to 4.5 using acetic acid. Then, the protein is loaded onto the SP Sepharose FF column, which has been equilibrated with 25 mM sodium acetate buffer (Buffer A) containing 1 mM EDTA at pH 4.5, at a binding density of 40-50 mg / ml to the resin. After loading, the column is washed with 2-3 column volumes of equilibration buffer. Then, the column is washed with a stepwise gradient using 20% ​​Buffer B (Buffer B: Buffer A + 0.5 M NaCl). Elution is performed using a stepwise gradient of 70% Buffer B. Then, the eluate is immediately diluted with water in a 1:1 ratio to prevent protein aggregation. The eluted protein is then subjected to buffer exchange using 25 mM Tris buffer (pH 8.0) with a 3 kDa membrane.

[0180] Anion exchange chromatography: After changing the buffer, the protein is loaded onto a Source 30Q resin, the column is equilibrated with 25 mM Tris buffer (pH 8.0), and then the protein solution is loaded. After loading, the column is washed with equilibrium buffer. Elution is then performed by flowing a linear gradient of elution buffer containing 25 mM Tris and 0.5 M NaCl, pH 8.0, in a column volume of 15. The final eluate is then re-buffered with TBS buffer (50 mM Tris buffer, 150 mM NaCl, pH 7.8). The eluted protein is analyzed for purity and concentration by HPLC.

[0181] Array summary [ka]

[0182] The embodiments of the present invention are as follows: 1. A method for preparing recombinant lectin protein, comprising the step of expressing a recombinant lectin protein encoded by a recombinant lectin gene in a host cell culture medium, wherein the cell expression is carried out under conditions such that the cells have a doubling time of 160 minutes or less.

[0183] 2. The method according to 1, wherein cell expression is carried out under conditions such that the cells have a doubling time of at least 100 minutes.

[0184] 3. Expression is carried out at a temperature of 22°C or lower, according to the method described in 1 or 2.

[0185] 4. Expression is carried out at a temperature of at least 15°C, according to any one of the methods described in items 1 to 3.

[0186] 5. The method according to any one of claims 1 to 4, comprising the step of culturing host cells, wherein the culturing includes a growth period (during which the host cells are grown before protein expression); and an expression period (during which protein expression takes place), wherein the growth period is carried out at a temperature higher than the temperature at which the expression period takes place.

[0187] 6. The growth period is carried out at a temperature of at least 25°C and 40°C or lower, as described in 5.

[0188] 7. The method according to 5 or 6, wherein the temperature from the growth period to the expression period is lowered for a period of at least 4 hours and no more than 7 hours.

[0189] 8. The method according to any one of items 1 to 7, wherein the expression of recombinant lectin protein is initiated by the addition of an inducer to the culture medium.

[0190] 9. The inducing substance is added to the culture medium at a concentration of at least 0.1 mM and no more than 0.5 mM, according to method 8.

[0191] 10. The derivative is an IPTG, as described in 8 or 9.

[0192] 11. The method described in any one of items 1 to 10, wherein the expression is carried out for at least 10 hours.

[0193] 12. The method according to any one of claims 1 to 11, wherein the carbon source is added to the culture medium at a rate of 2 g / L / h or less, and / or the nitrogen source is added to the culture medium at a rate of 1.5 g / L / h or less.

[0194] 13. The carbon source is added to the culture medium at a rate of at least 0.5 g / L / h in 12 different ways.

[0195] 14. The nitrogen source is added to the culture medium at a rate of at least 0.4 g / L / h, by method 12 or 13.

[0196] 15. The carbon source comprises or consists of glycerol, according to any one of items 12 to 14.

[0197] 16. Recombinant lectin proteins are i) Sequence ID 1; ii) Sequence ID 3; iii) Sequence ID 4; or iv) Amino acid sequences having at least 60% homology to i), ii), or iii) The method according to any one of items 1 to 15, comprising an amino acid sequence selected from the following.

[0198] The method according to 16, wherein the amino acid sequence of 17.iv) is an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% homology to i), ii), or iii).

[0199] 18. The method according to any one of claims 1 to 17, further comprising the step of isolating a crude recombinant lectin protein after the step of expressing a recombinant lectin protein.

[0200] 19. The method according to 18, further comprising the step of purifying a crude recombinant lectin protein.

[0201] 20. Purification of crude protein by the method in 19, comprising at least one chromatographic step.

[0202] 21. The method according to 20, wherein at least one chromatography step includes anion exchange chromatography and / or cation exchange chromatography.

[0203] 22. The method according to 20 or 21, wherein at least one chromatography step includes hydrophobic interaction chromatography.

[0204] 23. Purification of crude recombinant lectin protein is performed according to any one of items 19 to 22, including a filtration step.

[0205] 24. The host cell is Escherichia coli, as described in any one of items 1 to 23.

[0206] 25. The host cell comprises an expression construct containing a recombinant lectin gene, as described in any one of items 1 to 24.

[0207] 26. The host cell having a volume of at least 10 L, according to the method of any one of items 1 to 25.

[0208] 27. Expression is carried out in an industrial fermenter, as described in 26.

Claims

1. A method for preparing a recombinant lectin protein having improved cleavage of the initiation methionine compared to the lectin protein of SEQ ID NO: 2, the method comprising the steps of expressing a recombinant lectin protein encoded by a recombinant lectin gene in a host cell culture medium and isolating and purifying the recombinant lectin protein, The expression process includes a proliferation period (a period during which host cells are proliferated before protein expression), an induction period (a period during which an inducer is added to the culture medium), and an expression period (a period during which protein expression takes place). a) The host cell is E. coli, and the expression is carried out under conditions where the cell has a doubling time of 100–160 minutes, allowing sufficient time for methionine cleavage by methionine aminopeptidase (MAP) to increase the yield of recombinant lectin protein that does not contain methionine. b) Recombinant lectin protein expression is carried out at a temperature of 15–22°C, and the carbon-to-nitrogen ratio is maintained at 3:1–6:1 during the expression period. Recombinant lectin proteins include: i. Sequence ID 1; ii. Sequence ID 3; iii. Sequence ID 4; or iv. Amino acid sequences having at least 90%, 95%, 97%, 98%, or 99% homology to i, ii, or iii. Selected from, c) Perform the aforementioned expression period for 10 hours; d) During the expression period, add a carbon source to the culture at a rate of 0.5 to 2 g / L / h; e) During the expression period, add a nitrogen source to the culture at a rate of 0.4 to 1.5 g / L / h; f) During the induction period, the concentration of the induction substance shall be at least 0.1 mM and no more than 0.5 mM; the isolation and purification step shall be: g) Purify the crude recombinant lectin protein isolated from the host cell pellet by at least one chromatographic step; The method.

2. The method according to claim 1, wherein the host cell culture medium has a volume of at least 10 L.

3. The method according to any one of Claims 1 to 2, wherein the growth period is carried out at a temperature higher than the temperature at which the expression period is carried out, and the temperature is lowered over a period of at least 4 hours and no more than 7 hours from the growth period to the expression period.

4. The method according to claim 3, wherein the expression period is induced by the addition of an inducer substance at a concentration of at least 0.1 mM and 0.5 mM or less, and the inducer substance is IPTG (isopropyl thio-galactopyranoside).

5. The method according to claim 3 or 4, wherein the expression period is induced by the addition of an inducing substance when the absorbance of the culture medium is at least 25 and 40 or less.

6. The method described above is Furthermore, the method includes cloning a recombinant lectin gene into an expression vector and inserting the expression vector into a host cell. The growth period is carried out at a temperature of 25°C to 40°C, and the expression period is carried out at a temperature of at least 15°C and 22°C or lower, and the carbon-to-nitrogen ratio is maintained at 3:1 to 6:1 during the expression period. The method according to any one of claims 1 to 5, characterized in that the isolation is carried out by centrifugation followed by destruction of the cell surface.

7. The method according to claim 6, wherein the carbon source is added to the culture medium at a rate of at least 0.5 g / L / h and 2 g / L / h or less during the expression period, and the carbon source is glucose or glycerol, and the nitrogen source is added to the culture medium at a rate of at least 0.4 g / L / h and 1.5 g / L / h or less during the expression period, and the nitrogen source is tryptone, peptone, or yeast extract.

8. The method according to claim 6, wherein the purification of the crude recombinant lectin protein comprises at least one chromatography step selected from anion exchange chromatography, cation exchange chromatography and hydrophobic interaction chromatography.

9. The method according to claim 6, wherein the purification of the crude recombinant lectin protein includes a filtration step.

10. The isolation and purification step is a) A step of first purifying the crude recombinant lectin protein by anion exchange chromatography to obtain a first purified eluate; (i) A step of purifying the first purified eluate by hydrophobic interaction chromatography to obtain a second purified eluate; (c) A step of purifying the second purified eluate by cation exchange chromatography to obtain a third purified eluate; (e) A step of further purifying the first, second, or third purified eluate by anion exchange chromatography to obtain a fourth purified eluate; and (e) A step to obtain a purified recombinant lectin protein isolate by replacing the buffer of the fourth purified eluate by diafiltration. Features including, The method according to any one of claims 1 to 9.

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