A method for soluble expression and purification of recombinant human interleukin-2 or / and its mutants in prokaryotic system

Abstract

The application provides a method for soluble expression and purification of recombinant human interleukin-2 or / and mutants thereof in a prokaryotic system, which utilizes a method for tandem fusion of two or more Sumo sequences, realizes soluble expression of human IL-2 in an E. coli expression system, avoids formation of inclusion bodies of IL-2 expressed by a traditional E. coli system, avoids expression of IL-2 and IL-2M in the form of inclusion bodies without structure and activity, avoids a process of IL-2 protein renaturation in the downstream, and realizes efficient purification of rhIL-2 and IL-2M proteins from a complex bacterial broken supernatant system through a simple chromatography step. The method has the characteristics of simple operation, suitability for scale-up, short cycle, high purity, stable process and the like.

Description

Technical Field

[0001] This invention relates to the field of human interleukin-2 preparation technology, specifically to a method for the soluble expression and purification of recombinant human interleukin-2 or / and its mutants in a prokaryotic system. Background Technology

[0002] Human interleukin-2 (hIL-2), also known as T-cell growth factor, is one of the many important cytokines in the human body. Natural human interleukin-2 is composed of 133 amino acids, with a theoretical molecular weight of approximately 15.4 kDa and a theoretical isoelectric point of 6.7. Human IL-2 contains a high proportion of leucine (16.5%), one glycosylation site (Thr3), and three cysteine ​​residues. The disulfide bond formed between Cys58 and Cys105 is crucial for the structural stability and biological activity of IL-2. Human IL-2 mediates physiological signals and exerts its immunomodulatory function by binding to three receptors: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγc (CD132). IL-2Rα is the specific receptor for IL-2 (Kd = ~10⁻⁸ M); IL-2Rβ is a common receptor for IL-2 and IL-15; and IL-2Rγc is a common receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 binding to IL-2Rα alone cannot mediate effective physiological signals. IL-2 can effectively mediate physiological signal transduction with both dimerizing receptors (IL-2Rβ and IL-2Rγc, Kd = ~10⁻⁹ M) and trimerizing receptors (IL-2Rα, IL-2Rβ, and IL-2Rγc, Kd = ~10⁻¹¹ M).

[0003] Chiron's rhIL-2 (Aldesleukin; Proleukin®) was approved by the FDA in 1992 for the treatment of renal cell carcinoma and in 1998 for the treatment of melanoma. Aldesleukin is a structurally optimized version of human IL-2. Because it is expressed in *E. coli*, there is no glycosylation at the Thr3 position, and the N-terminal Ala is also deleted. Furthermore, to avoid or reduce the proportion of disulfide bond mispairing during expression and renaturation in *E. coli*, Aldesleukin uses a Ser125 mutation to replace Cys125 (C125S). Compared to native human IL-2, Aldesleukin lacks the N-terminal alanine residue, and the cysteine ​​residue at 125 is replaced with serine. However, its in vitro bioactivity is not significantly different from that of native IL-2. Due to the lack of glycosylation, Aldesleukin has a slightly shorter in vivo half-life than native human IL-2, approximately 13–85 minutes.

[0004] Human IL-2 contains a high proportion of leucine (16.5%), exhibiting strong hydrophobicity. Its recombinant expression and purification have always been a technical challenge in the pharmaceutical industry. Prokaryotic expression systems, such as those using *E. coli*, offer advantages such as low cost, short culture period, and high expression levels, making them one of the most commonly used recombinant expression systems for exogenous proteins. Chiron Corporation expressed Aldesleukin as inclusion bodies using an *E. coli* expression system. Then, through urea denaturation, dissolution with sodium dodecylbenzenesulfonate and 2-butanol, reducing reduction, and oxidative oxidation to promote disulfide bond formation, structurally refolded IL-2 was obtained. Further purification using gel filtration chromatography and reversed-phase high-performance liquid chromatography yielded IL-2 with a purity exceeding 98% (US4569790, EP0470586B1, Purified recombinant interleukin-2 compositions, Chiron Corporation, Emeryville, California 94608 (US), 1985). Malcolm P. et al. successfully expressed IL-2 in Escherichia coli as inclusion bodies. The expression was denatured in 6M guanidine hydrochloride and 10mM DTT in pH 8.5 buffer. The expression was purified by gel filtration chromatography under denaturing and reducing conditions. After dilution and refolding and auto-oxidation refolding, the expression was finally purified by reversed-phase high-performance liquid chromatography to obtain IL-2 with a yield of 30% and a purity of more than 95% (Weir MP, Sparks J. Purification and renaturation of recombinant human interleukin-2. Biochem J. 1987 Jul 1;245(1):85-91.).Esfandiar S. et al. fused a His-tag (6xHistine) to the N-terminus of IL-2 and expressed it in E. coli. The His-tag IL-2 was expressed in E. coli as inclusion bodies. After denaturation with 8 M urea and 10 mM mercaptoethanol, combined with inclusion body refolding and Ni2+ metal chelate affinity chromatography, His-IL-2 with a purity higher than 97% was obtained. The His-IL-2 obtained had similar activity to IL-2 (Esfandiar S, Hashemi-Najafabadi S, Shojaosadati SA, Sarrafzadeh SA, Pourpak Z. Purification and refolding of Escherichia coli-expressed recombinant human interleukin-2. Biotechnol ApplBiochem. 2010 Apr 14;55(4):209-14.). Boozarpour, S. et al. fused mouse IL-2 (mIL-2) with the thioreductase protein (TrxA) gene, constructed the pET-32a expression vector, and expressed it in E. coli BL21 (DE3). They successfully expressed most of mIL-2 in E. coli in a soluble form. However, when human IL-2 was fused with TrxA using the same method, TrxA-hIL-2 was almost entirely expressed in an insoluble form (Boozarpour, S., Sadeghizadeh, M., Shokrgozar, MA, Hosseinkhani, S., Shojaosadati, SA, Gharavi, S., Ahangari, G., & Ranjbar, B. (2010). Bacterial overexpression of the human interleukin-2 in insoluble form via the pET Trx fusion system. Iranian Journal of Biotechnology, 8, 270-274.). While expressing human IL-2 using prokaryotic expression systems (commonly E. coli) has advantages such as low culture cost, short cycle, and high expression level, there are still problems such as IL-2 often being expressed in the form of inclusion bodies, requiring denaturation and refolding of human IL-2 in downstream processes. The refolding of human IL-2 inclusion bodies often results in extremely low refolding yield and is extremely difficult to process.

[0005] To attempt the soluble expression of human IL-2 and its mutants, Liu Y. et al. constructed the pPIC9K-MhIL-2 expression vector and transformed it into Pichia pastoris GS115. Positive clones were obtained through G418 pressure selection, and the human IL-2 mutant (MhIL-2 (L18M / L19S / C125A)) was successfully expressed in soluble form through fermentation at an expression level of approximately 100 mg / L. After purification by cation exchange chromatography and gel filtration chromatography, MhIL-2 (L18M / L19S / C125A) protein with a purity higher than 90% and a yield higher than 20% was obtained (Liu Y, Xiao XY, Sun M, Hu YH, Ou-Yang KQ, Cai SX, Hua ZC. Expression and purification of a mutant of humaninterleukin-2 in Pichia pastoris. Appl Biochem Biotechnol. 2006 Apr;133(1):77-86.). Similarly, Ahmed N. et al. constructed the human IL-2 gene in the pPICZα vector and transformed this vector into Pichia pastoris GS115. After resistance selection, positive clones of yeast were obtained, and soluble expression of human IL-2 was successfully achieved through fermentation, with an expression level as high as approximately 210 mg / L. Through screening for IL-2 affinity ligands, affinity chromatography using Blue SA P6XL as the affinity ligand successfully purified IL-2 to obtain hIL-2 (C125S) protein with a purity higher than 97% and a yield of nearly 55% (Ahmed, N., Khan, MA, Shahid, N., Nasir, IA, & Zafar, AU (2011). One steppurification of biologically active human interleukin-2 protein produced in yeast (Pichia Pastoris). African Journal of Biotechnology, 10, 15170-15178.). Expressing recombinant rhIL-2 protein through a yeast expression system still faces challenges such as long culture cycles, high culture costs, and complex downstream preparation processes.

[0006] In addition, Roche has produced human IL-2 by culturing induced human malignant tumor cells, such as human leukemia and lymphoma cells (H33HJ-Jal cell line). During the culture process, T cell mitogens (such as PHA) stimulate the cells to secrete IL-2 into the culture supernatant. Human IL-2 is then separated by anion exchange chromatography (CM medium) and reversed-phase high-performance liquid chromatography (US5597901, Homogeneous human interleukin 2, Hoffmann-La Roche Inc. (Nutley, NJ), 1997). However, producing human IL-2 protein through human tumor cell culture also has drawbacks, including long production cycles, extremely high culture costs, and extremely low protein expression levels. Summary of the Invention

[0007] The purpose of this invention is to provide a method for soluble expression and purification of recombinant human interleukin-2 and / or its mutants, addressing the shortcomings of existing technologies and the aforementioned difficulties. The downstream process does not involve inclusion body denaturation and renaturation steps, the culture process is simple and extremely low-cost, the expression cycle of hIL-2 or its mutants is short, the downstream purification steps are simple, and the prepared hIL-2 or its mutant protein has the correct structure and maintains good in vitro activity.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for soluble expression and efficient purification of recombinant human interleukin-2 and / or its mutants, comprising the following steps:

[0010] Step 1: Splice the gene sequence of human interleukin-2 or / and human interleukin-2 mutant with the 3' end of the gene sequence of two or more small ubiquitin-related modifiers to obtain the gene sequence of His-(Sumo)n-IL-2 (n is ≥2, a natural number) or / and its mutant.

[0011] Step 2: Insert the gene sequence of His-(Sumo)n-IL-2 or / and its mutant obtained in Step 1 into the prokaryotic expression plasmid vector, and then transform the plasmid vector into prokaryotic competent cells;

[0012] Step 3: Select dominant growth colonies by resistance plating, expand culture, and obtain the fusion protein His-(Sumo)n-IL-2 or / and its mutant by intervening in the promoter to transcribe and translate the target gene.

[0013] Step 4: Lyse the bacterial cells, centrifuge to obtain the supernatant, and purify the supernatant to obtain high-purity recombinant His-(Sumo)n-IL-2 or / and its mutant fusion protein by the first metal chelate chromatography. After removing the His-(Sumo)n fusion sequence by enzyme excision, the protein is then subjected to a second metal chelate chromatography to obtain recombinant human interleukin-2 or / and its mutant.

[0014] Based on human IL-2 (UniProtKB - P60568, with or without the N-terminal alanine (Ala) and / or proline (Pro)), the rhIL-2 gene sequence (SEQ ID NO: 1) was obtained through codon preference optimization in *E. coli*. Specific (single or multiple site) amino acid mutations can then be performed to derive rhIL-2 mutants, such as C125S and C125A, but not limited to these. Using PCR technology, with the aid of specific restriction endonucleases and primers, the gene sequences of hIL-2 and its mutant (IL-2m) can be spliced ​​to the 3' end of two or more small ubiquitin-associated modifier (Sumo) tandem gene sequences to obtain the His-(Sumo)n-IL-2 (n≥2) gene sequence. This His-(Sumo)n-IL-2 (n≥2) gene sequence is then inserted into an *E. coli* expression plasmid vector using PCR, and the plasmid is subsequently transformed into competent *E. coli* cells. Dominant colonies were selected by resistance plating and expanded culture. The target gene was transcribed and translated to obtain the fusion protein His-(Sumo)n-IL-2 (n≥2) by promoter intervention. After induction, the bacterial cells were harvested, lysed, and centrifuged to obtain the supernatant. This supernatant was then purified by metal chelate affinity chromatography (adsorption-elution mode) to obtain His-(Sumo)n-IL-2 (n≥2). The His-(Sumo)n (n≥2) fusion protein was then removed by ULP1 restriction enzyme digestion, followed by further purification by metal chelate affinity chromatography (flow-through mode) to finally obtain hIL-2 or hIL-2-M protein.

[0015] This invention provides a method for the soluble expression and efficient purification of recombinant human interleukin-2 and / or its mutants in a prokaryotic system. First, the gene sequences of rhIL-2 and its mutant IL-2M are spliced ​​to the 3' end of the His-(Sumo)n (n≥2) gene sequence, inserted into a prokaryotic expression vector, and transformed into competent prokaryotic cells. Then, the fusion protein of His-(Sumo)n (n≥2) with IL-2 and the IL-2 mutant is expressed in a specific prokaryotic system. In its soluble form, it is highly expressed intracellularly. The fusion proteins of His-(Sumo)n (n≥2) with IL-2 and IL-2 mutants do not require inclusion body refolding. The His-(Sumo)n-IL-2 and IL-2 mutant fusion proteins are obtained via a one-step metal chelate chromatography (adsorption-elution mode). After removing the His-(Sumo)n fusion sequence by ULP1 enzyme digestion, recombinant human IL-2 and its mutants are obtained via a further one-step metal chelate chromatography (flow-through mode). This method utilizes the tandem fusion of two or more Sumo sequences to achieve soluble expression of human IL-2 in an E. coli expression system, avoiding the inclusion body formation and refolding process required in traditional E. coli IL-2 expression systems. It features simple operation, suitability for large-scale scaling, short cycle time, high purity, and stable process.

[0016] By utilizing the soluble high expression of rhIL-2 and its mutant (IL-2M) protein in prokaryotic Escherichia coli, the formation of inactive and structureless inclusion bodies during the expression of the highly hydrophobic rhIL-2 and its mutant in prokaryotic cells is avoided, and the subsequent downstream processes do not require inclusion body refolding.

[0017] The rhIL-2 and IL-2M fusion proteins were directly and solublely expressed in a prokaryotic Escherichia coli system. The N-terminus of the fusion protein contained two or more Sumo or Sumo-modified (His-(Sumo)n, n≥2) fusion protein sequences and affinity purification tags to promote the correct folding of rhIL-2 and IL-2M fusion proteins in the prokaryotic expression system.

[0018] Fusion proteins (His-(Sumo)n-IL-2 or His-(Sumo)n-IL-2M) were efficiently purified from a complex system of E. coli bacterial lysate supernatant using a one-step metal chelate chromatography method (adsorption-elution mode). After removing the His-(Sumo)n fusion sequence by ULP1 restriction enzyme, the fusion proteins were further purified using a one-step metal chelate chromatography method (flow-through mode) to obtain rhIL-2 or IL-2M proteins. The prepared rhIL-2 and IL-2M proteins exhibited the correct spatial structure and good biological activity.

[0019] Furthermore, the more sumo units n values ​​there are, the stronger the expression-promoting effect.

[0020] Furthermore, in step 1, the amino acid sequence of human interleukin-2 and / or the amino acid sequence of human interleukin-2 mutant are sequentially subjected to gene sequence conversion and E. coli host preference codon optimization to obtain the gene sequence of human interleukin-2 and / or human interleukin-2 mutant.

[0021] Furthermore, the sequence of human interleukin-2 is the complete human IL-2 protein (UniProtKB - P60568), or it may have one (proline, Pro) or several amino acid residues missing or replaced at its N-terminus or in the sequence.

[0022] Furthermore, human IL-2 mutants (IL-2M) are structural equivalents of rhIL-2. Without changing the main spatial structure of rhIL-2 (i.e., antiparallel quadruple helical bundles), mutants that replace, delete, or insert one or more amino acids at any site in the primary structure amino acid sequence can be regarded as structural equivalents of rhIL-2, i.e., IL-2M.

[0023] Furthermore, the gene sequence of human interleukin-2 is an optimized E. coli preferred codon, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0024] Furthermore, the gene sequence of the IL-2 mutant is a gene sequence obtained by substituting one or more amino acid codons based on the gene sequence of human interleukin-2, for example (SEQ ID NO: 2; SEQ ID NO: 3).

[0025] Furthermore, in His-(Sumo)n-IL-2, His-(Sumo)n is a tandem of two or more Sumo protein sequences, n≥2, and is a natural number.

[0026] Furthermore, the Sumo protein sequence is a small ubiquitin-related modifier, the most representative of which is the sequence of the ubiquitin-like protein SMT3 (UniProtKB - Q12306) from the species Saccharomyces cerevisiae (strain ATCC 204508 / S288c), whose N-terminus is fused with 6xHis protein sequence as shown in (SEQ ID NO: 8), or a Sumo modified form by replacing or mutating one or more amino acid residues in its primary sequence.

[0027] Furthermore, the tandem pattern of two or more Sumo protein sequences can be the tandem of multiple Sumo repeat sequences, the tandem of multiple Sumo modifiers, or the tandem of Sumo and Sumo modifiers. One representative tandem pattern is His-Sumo modifier-Sumo-IL2.

[0028] Furthermore, Sumo mutants replace or mutate one or more amino acid residues in their protein sequence, especially one or more amino acid residues of the five amino acids EQIGG at the C-terminus. A typical Sumo mutant has the EQIG sequence at the C-terminus.

[0029] Furthermore, when the Sumo mutant is fused with other protein sequences at its C-terminus, the ULP1 enzyme cannot recognize the C-terminal sequence of the Sumo mutant, making it difficult to cleave it.

[0030] Furthermore, the prokaryotic competent cells are *Escherichia coli* or *Bacillus subtilis*. Characteristics of prokaryotic competent cell hosts include short doubling cycles, simple culture medium requirements, and high recombinant protein expression yields. *Escherichia coli*, such as *E. coli* BL21 DE3, is preferred, and even more preferred are *E. coli* BL21 Origami (DE3) and Rosetta-gami (DE3) pLysS hosts, but these are not limited to these.

[0031] Furthermore, the plasmid vector is a pET series plasmid or a pBV series plasmid. The prokaryotic expression vector is a plasmid capable of self-replication and amplification, initiating transcription and translation in prokaryotic bacteria. Preferably, the plasmid vector is the pET-32a plasmid.

[0032] Furthermore, when n is 2, the fusion protein His-(Sumo)2-IL-2 has the amino acid sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 5.

[0033] Furthermore, in step 3, the operation of initiating transcription of the plasmid carrying the target gene (His-(Sumo)n-IL-2) includes, but is not limited to, adding a certain concentration of inducing agent for the intervention promoter, raising or lowering the culture temperature, or using self-induction in the culture medium. A commonly used and representative method for initiating transcription of the target gene in the plasmid by intervention promoter is to add IPTG or use lactose and other lactose structural analogs, such as the T7 promoter type of pET series plasmids. Another common and representative method for promoting soluble expression of the target gene is to lower the culture temperature during induction, such as 20-30℃.

[0034] Furthermore, in step 4, the first metal chelation chromatography is in adsorption-elution mode; the second chelation chromatography is in flow-through mode.

[0035] Furthermore, in the first metal chelate chromatography and the second metal chelate chromatography, the solid-phase chromatography packing material is a type of chromatography packing material that can be tandemly fused with three or more histidine residues for affinity binding and adsorption; the solid-phase chromatography microsphere packing material chelates divalent metal ions, including nickel ions, copper ions, zinc ions and cobalt ions, by deriving chelating groups.

[0036] Furthermore, the specific operational steps of step 4 are as follows:

[0037] Step 41: After expression, the bacterial cells are resuspended in resuspension buffer and then lysed to release intracellular proteins into extracellular resuspension buffer. The bacterial lysis supernatant is then centrifuged.

[0038] Step 42: The supernatant obtained in step 41 is purified by nickel metal chelate chromatography. After the sample flows through the chromatography packing material, His-(Sumo)n-IL-2 or / and the mutant target protein are adsorbed on the chromatography packing material. After appropriate rinsing, the protein is finally eluted with imidazole elution buffer.

[0039] The protein samples obtained in steps 43 and 42 were subjected to imidazole removal and buffer replacement. The buffer consisted of 50 mM PB and 100 mM arginine at pH 7.0. Then, the His-(Sumo)n fusion protein was digested with ULP1 enzyme to release free IL-2 and / or mutants. The digestion time was 1 to 24 hours, and the digestion temperature was 4 to 30°C. The ratio of ULP1 enzyme used was 1:10 to 10000.

[0040] Step 44: The enzyme-digested sample obtained in step 43 is subjected to nickel metal chelate chromatography purification again. ULP1 enzyme, His-(Sumo)n protein and His-(Sumo)n-IL-2 fusion protein that has not been effectively digested are adsorbed onto the chromatographic packing material. The target protein IL-2 or / and its mutant flows through the chromatographic column. Finally, the flow-through is collected, concentrated, and the medium is changed to obtain recombinant human interleukin-2 or / and its mutant.

[0041] The sequences involved in the instruction manual are as follows:

[0042] SEQ ID NO:1

[0043] The rhIL-2 gene sequence was optimized using E. coli preferred codons.

[0044] GCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTTGTCAGAGCATTATTAGCACCCTGACC。

[0045] SEQ ID NO:2

[0046] The rhIL-2 gene sequence after one or several amino acid codon substitutions

[0047] GCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTAGCCAGAGCATTATTAGCACCCTGACC。

[0048] SEQ ID NO:3

[0049] The rhIL-2M gene sequence after one or several amino acid codon substitutions

[0050] ACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTGCAAATTCTATATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTAGCCAGAGCATTATTAGCACCCTGACC。

[0051] SEQ ID NO:4

[0052] 6×His-(Sumo)2-rhIL-2 amino acid sequence

[0053] HHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT。

[0054] SEQ ID NO:5

[0055] His-Sumo-Sumo-rhIL-2 gene sequence

[0056]

[0057] SEQ ID NO:6

[0058] rhIL-2 amino acid sequence

[0059] TSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT.

[0060] SEQ ID NO:7

[0061] rhIL-2M amino acid sequence

[0062] TSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTCKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT.

[0063] SEQ ID NO:8

[0064] His-Sumo amino acid sequence

[0065] HHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.

[0066] SEQ ID NO:9

[0067] His-Sumo-rhIL-2 amino acid sequence

[0068] HHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT。

[0069] SEQ ID NO:10

[0070] His-Sumo-rhIL-2 gene sequence

[0071] CATCATCACCATCATCATAGCGATAGCGAAGTTAATCAAGAAGCCAAACCGGAAGTTAAGCCGGAAGTGAAACCTGAAACACATATTAACCTGAAAGTGAGTGATGGTAGCAGCGAGATCTTTTTCAAAATCAAAAAGACCACACCGCTGCGTCGTCTGATGGAAGCATTTGCAAAACGTCAGGGTAAAGAAATGGATAGCCTGCGTTTTCTGTATGATGGTATTCGTATTCAGGCAGATCAGACACCGGAAGATCTGGATATGGAAGATAACGATATTATCGAAGCACATCGTGAGCAGATTGGTGGTGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTAGCCAGAGCATTATTAGCACCCTGACC。

[0072] SEQ ID NO:11

[0073] Amino acid sequence of His-(Sumo)2

[0074] HHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.

[0075] This invention provides a method for the soluble expression and efficient purification of recombinant human interleukin-2 and / or its mutants in a prokaryotic system. First, the gene sequences of rhIL-2 and its mutant IL-2M are spliced ​​to the 3' end of the His-(Sumo)n (n≥2) gene sequence, inserted into a prokaryotic expression vector, and transformed into competent prokaryotic cells. Then, the fusion protein of His-(Sumo)n (n≥2) with IL-2 and the IL-2 mutant is expressed in a specific prokaryotic system. In the soluble state, it is highly expressed intracellularly; the fusion protein of His-(Sumo)n (n≥2) with IL-2 and IL-2 mutant does not need to be renatured by inclusion bodies. The His-(Sumo)n-IL-2 and IL-2 mutant fusion protein are obtained by one-step metal chelate chromatography (adsorption-elution mode). After the His-(Sumo)n fusion sequence is removed by ULP1 enzyme digestion, the recombinant human IL-2 and its mutant are obtained by one-step metal chelate chromatography (flow-through mode). This method utilizes the tandem fusion of two or more Sumo sequences to achieve soluble expression of human IL-2 in an E. coli expression system. This avoids the formation of inclusion bodies in traditional E. coli IL-2 expression systems, prevents the expression of IL-2 and IL-2M in the form of unstructured and inactive inclusion bodies, and avoids the downstream IL-2 protein refolding process. Furthermore, it efficiently purifies rhIL-2 and IL-2M proteins from a complex bacterial lysate supernatant system through a simple chromatographic step. It features simple operation, suitability for large-scale scaling, short cycle time, high purity, and stable process. Attached Figure Description

[0076] Figure 1 This is a schematic diagram of the characteristic structures of His-Sumo-rhIL-2 and His-Sumo-rhIL-2M plasmids in Example 1, along with their nucleic acid electrophoresis identification.

[0077] Figure 2 This is a schematic diagram of the characteristic structures of His-(Sumo)2-rhIL-2 and His-(Sumo)2-rhIL-2M plasmids in Example 1, and a nucleic acid electrophoresis identification diagram.

[0078] Figure 3 This is the SDS-PAGE identification image of His-Sumo-rhIL-2 and His-Sumo-IL-2M in Example 2.

[0079] Figure 4 This is an SDS-PAGE identification image of His-2Sumo-rhIL-2 and His-2Sumo-IL-2M soluble expression in Example 2.

[0080] Figure 5 These are the metal chelate chromatography purification chromatograms and electrophoretic identification results of His-2Sumo-rhIL-2 protein in Example 3. Figure 5 (a) in the figure is the result of metal chelation chromatography. Figure 5 (b) The purity results of SDS-PAGE characterization.

[0081] Figure 6 These are the chromatography patterns and electrophoretic identification images of His-2Sumo-IL-2M metal chelate chromatography purification in Example 3, wherein... Figure 6 (a) in the figure is the result of metal chelation chromatography. Figure 6 (b) The purity results of SDS-PAGE characterization.

[0082] Figure 7 These are the chromatographic patterns and electrophoretic identification images of the fusion protein after ULP1 digestion of IL-2 and IL-2M.

[0083] Figure 8 This is a comparison of the circular dichroism spectral structures of rhIL-2 and rhIL-2M prepared in Example 4.

[0084] Figure 9 This is a comparison of the fluorescence spectral structure characterization of rhIL-2 and rhIL-2M prepared in Example 4.

[0085] Figure 10 The results are obtained by high-performance gel filtration chromatography of rhIL-2 and rhIL-2M prepared in Example 4.

[0086] Figure 11 The results are from the in vitro cell proliferation (CTLL-4) experiment of rhIL-2 and rhIL-2 standard protein prepared in Example 4. Detailed Implementation

[0087] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Example 1

[0088] Construction of His-(Sumo)2-rhIL-2-pET-32a and His-(Sumo)2-rhIL-2M-pET-32a expression vectors

[0089] The His-(Sumo)2-rhIL-2-pET-32a and His-(Sumo)2-rhIL-2M-pET-32a expression vectors were obtained through two PCR experiments. First, the rhIL-2 amino acid sequence (133AA, C125S), amino acid sequence (SEQ ID NO: 6), and the rhIL-2M amino acid sequence (133AA, F42C, C125S), amino acid sequence (SEQ ID NO: 7), were subjected to gene sequence conversion and E. coli host-preferred codon optimization to obtain SEQ ID NO: 1 and its mutant sequence. The rhIL-2 and rhIL-2M gene sequences were then synthesized and inserted into the His-Sumo-pET-32a plasmid vector (a plasmid delivered by a gene synthesis company). The amino acid sequence of His-Sumo is (SEQ ID NO: 8). Using rhIL-2-pET-30a and rhIL-2M-pET-30a as templates, upstream and downstream primers were designed, and the rhIL-2 gene sequence was obtained by PCR amplification.

[0090] The PCR reaction conditions were: 98℃ pre-denaturation for 2 min, 98℃ for 10 s, 54℃ for 15 s, 72℃ for 10 s, 35 cycles, followed by an extension at 72℃ for 5 min. After identification of the PCR products by 2% agarose gel electrophoresis, the rhIL-2 and rhIL-2M gene sequences were inserted into the His-Sumo-pET-32a vector. Specific procedures included: designing upstream and downstream primers to amplify the His-Sumo-pET-32a plasmid; homologous recombination of the fragment with the vector yielded the His-Sumo-IL-2-pET-32a and His-Sumo-rhIL-2M-pET-32a recombinant plasmids. The amino acid sequence of His-Sumo-rhIL-2 is (SEQ ID NO: 9), and its corresponding gene sequence is (SEQ ID NO: 10). PCR reaction conditions were as follows: 98℃ pre-denaturation for 5 min, 98℃ for 10 s, 48℃ for 15 s, 72℃ for 90 s, 35 cycles, followed by an extension at 72℃ for 5 min. PCR products were identified by 0.7% agarose gel electrophoresis. The His-Sumo-rhIL-2-pET-32a and His-Sumo-IL-2M-pET-32a recombinant plasmids were transformed into DH5α competent cells and cultured on Amp+ resistant solid LB medium. Single colonies were selected and activated on LB medium at 37℃ for 16 h. Plasmids were then extracted, and plasmids with correct sequencing results were selected for subsequent experiments.

[0091] upstream primer sequence for rhIL-2 gene PCR amplification:

[0092] 5'-CGTGAGCAGATTGGTGGTACCAGCAGCAGCACCAAAAAAAC-3';

[0093] Downstream primer sequence for rhIL-2 gene PCR amplification:

[0094] 5'-GAATTCGGATCCGATGATATCTTAGGTCAGGGTGCTAATAATGC-3';

[0095] pET-32a-His-Sumo plasmid PCR amplification upstream primer sequence:

[0096] 5'-GATATCATCGGATCCGAATTCG-3';

[0097] pET-32a-His-Sumo plasmid PCR amplification downstream primer sequence:

[0098] 5'-GTACCACCAATCTGCTCACG-3';

[0099] The second step, PCR, involved inserting the gene sequence containing the Sumo fragment into the correctly sequenced His-Sumo-rhIL-2-pET-32a and His-Sumo-rhIL-2M-pET-32a plasmid vectors. Both the fragment and the vector were ~3000 bp. Using His-Sumo-rhIL-2-pET-32a and His-Sumo-rhIL-2M-pET-32a as templates, upstream and downstream primers were designed, and the results were obtained through PCR amplification.

[0100] The PCR reaction conditions were: 98℃ pre-denaturation for 2 min, 98℃ for 10 s, 61℃ for 15 s, 72℃ for 45 s, 35 cycles, followed by extension at 72℃ for 5 min. After identification of the PCR products by 1% agarose gel electrophoresis, the fragment carrying the Sumo gene sequence was inserted into the His-Sumo-rhIL-2pET-32a and His-Sumo-rhIL-2M-pET-32a vectors. Specific procedures: Upstream and downstream primers were designed to amplify the His-Sumo-pET-32a plasmid and the Sumo-rhIL-2-pET-32a and Sumo-rhIL-2-MpET-32a plasmids. After homologous recombination of the fragments with the vector, recombinant plasmids His-(Sumo)2-rhIL-2-pET-32a and His-(Sumo)2-rhIL-2M-pET-32a were obtained, with the amino acid sequence of His-(Sumo)2 being (SEQ ID NO: 11). The His-(Sumo)2-rhIL-2-pET-32a and His-(Sumo)2-rhIL-2M-pET-32a recombinant plasmids were transformed into DH5α competent cells and cultured in Amp+ resistant solid LB medium. Single colonies were screened. Single colonies were picked, activated in LB medium at 37℃ for 16 h, and plasmids were extracted. Plasmids with correct sequencing results were selected for subsequent experiments.

[0101] The plasmid was transformed into Origami B(DE3) competent cells to obtain Origami B(DE3) expression strains containing recombinant plasmids His-(Sumo)2-rhIL-2-pET-32a and His-(Sumo)2-IL-2M-pET-32a.

[0102] Upstream primer sequence for PCR amplification of the Sumo gene fragment:

[0103] 5'-CTATGAGAAAGCGCCACGCTTCCC-3';

[0104] Downstream primer sequence for amplifying the Sumo gene fragment:

[0105] 5'-ACCAATCTGCTCACGATGTGCTTC-3';

[0106] Upstream primer sequences for PCR amplification of the Sumo-rhIL-2 and Sumo-rhIL-2-M gene vector fragments:

[0107] 5'-CATCGGTGAGCAGATTGGTAGCGATAGCGAAGTTAATCAAGAAGCCAAAC-3';

[0108] Upstream primer sequences for PCR amplification of vectors containing Sumo-rhIL-2 and Sumo-rhIL-2-M genes:

[0109] 5'-CGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG-3'.

[0110] Among them, such as Figure 1 and Figure 2 As shown, Figure 1 The image shows schematic diagrams of the characteristic structures of His-Sumo-rhIL-2 and His-Sumo-rhIL-2M plasmids (top) and nucleic acid electrophoresis identification images (bottom); among them, Figure 1 (a) pET-32a-His-Sumo PCR amplification: Lane 1: 1 kb DNA Marker; Lane 2: pET-32a-His-Sumo. Figure 1 In (b) of the diagram, IL2 PCR amplification: Lane 1: 100 bp DNA Marker; Lane 2: IL2; Lane 3: IL-2M. Figure 2 The image shows schematic diagrams of the characteristic structures of His-(Sumo)2-rhIL-2 and His-(Sumo)2-rhIL-2M plasmids (top) and nucleic acid electrophoresis identification images (bottom). In the nucleic acid electrophoresis identification images, pET-32a-His-(Sumo)2-IL2 PCR amplification: Lane 1: 1kb DNA Marker; Lane 2: pET-32a-Sumo-IL-2M

[0111] Lane 3:pET-32a-Sumo-IL-2 Lane 4:pET-32a-His-Sumo. Example 2

[0112] Expression and identification of recombinant plasmids His-Sumo-rhIL-2-pET-32a, His-(Sumo)2-rhIL-2-pET-32a, His-Sumo-IL-2M-pET-32a, and His-(Sumo)2-IL-2M-pET-32a in Escherichia coli Origami B(DE3)

[0113] The recombinant engineered bacterial cultures containing His-(Sumo)2-rhIL-2-pET-32a and His-(Sumo)2-rhIL-2M-pET-32a recombinant plasmids were inoculated into 100 mL of LB medium containing ampicillin. The cultures were activated overnight (12 h) at 37°C and 200 rpm. The activated cultures were then inoculated at a ratio of 5% (v / v) into 1000 mL of LB medium containing 100 µg / mL ampicillin and cultured at 37°C and 200 rpm for approximately 4 h until OD (Organic Degradation). 600nm The concentration reached approximately 0.8. The culture temperature was lowered to 25°C, and after inducing recombinant gene expression on the plasmid for 12 hours, the culture was terminated. The bacterial cells were harvested by centrifugation, sonicated, and centrifuged again to obtain the supernatant. 15% SDS-PAGE analysis showed that His-Sumo-rhIL-2 and His-Sumo-IL-2M were mainly present in the centrifuged precipitate after bacterial lysis (inclusion bodies), while His-(Sumo)2-rhIL-2 and His-(Sumo)2-IL-2M were mainly present in the supernatant of bacterial lysis (soluble form), indicating that rhIL-2 is highly expressed primarily in the soluble form within the bacterial cell.

[0114] like Figure 3 and Figure 4 As shown, Figure 3 SDS-PAGE identification images of 6×His-Sumo-rhIL-2 and 6×His-Sumo-IL-2M expression forms (inclusion body expression) Figure 3 (a)pET-32a-6×His-Sumo-rhIL-2 expression in Lane 1: Protein Marker; Lane 2: Lane before induction; Lane 3: Lane after induction; Lane 4: Bacterial lysis supernatant; Lane 5: Bacterial lysis precipitate. Figure 3 (b) pET-32a-6×His-Sumo-IL-2M expression: Lane 1: Before induction; Lane 2: After induction; Lane 3: Bacterial lysis supernatant; Lane 4: Bacterial lysis precipitate; Lane 5: Protein Marker.

[0115] Figure 4SDS-PAGE identification diagram of soluble expression of 6×His-(Sumo)2-rhIL-2 and 6×His-(Sumo)2-IL-2M (soluble form expression); Figure 4 Identification of (a)pET-32a-6×His-(Sumo)2-rhIL-2 induction and soluble expression: Lane 1: rhIL-2 before induction; Lane 2: rhIL-2 after induction; Lane 3: rhIL-2 lysis supernatant; Lane 4: rhIL-2 lysis precipitate; Lane 5: Protein Marker. Figure 4 (b) pET-32a-6×His-(Sumo)2-rhIL-2M induction and soluble expression identification: Lane 1: rhIL-2M before induction; Lane 2: rhIL-2M after induction; Lane 3: rhIL-2M lysis supernatant; Lane 4: rhIL-2M lysis precipitate; Lane 5: Protein Marker. Example 3

[0116] rhIL-2 and IL-2-M purified by chromatography

[0117] rhIL-2 and rhIL-2M were lysed using 50 mM PB, 100 mM arginine, and pH 7.5. The lysed solution was centrifuged at 4°C and 12,000 rpm for 30 min, and the supernatant was collected for sample loading.

[0118] Affinity chromatography process (adsorption-elution mode): using Ni 2＋ -NTA column affinity chromatography purification. Before sample loading, the column was pre-washed with buffer B (50 mM PB, 100 mM arginine, 500 mM NaCl, 500 mM imidazole, pH 7.5) for 3-5 column volumes, then equilibrated with buffer A (50 mM PB, 100 mM arginine, 500 mM NaCl, pH 7.5) for 3-5 column volumes. After sample loading, the column was equilibrated again with buffer A for 3-5 column volumes, followed by elution with 35% and 100% buffer B sequentially. The elution products were collected and the purification results were identified by 15% SDS-PAGE.

[0119] Affinity chromatography procedure (flow-through elution mode): The purified product collected during affinity chromatography is digested with ULP1 enzyme. The collected buffer system containing the target protein is then replaced with buffer A (50 mM PB, 100 mM arginine, pH 7.5). The treated protein solution is then subjected to Ni... 2＋-NTA column affinity chromatography purification. Before sample loading, the column is pre-washed with buffer B (50 mM PB, 100 mM arginine, 500 mM NaCl, 500 mM imidazole, pH 7.5) for 3-5 column volumes, then equilibrated with buffer A for 3-5 column volumes. Sample is loaded and the flow-through is collected, then equilibrated with buffer A for 3-5 column volumes. Finally, the column is washed with 100% buffer B to remove contaminating proteins, and the elution product is collected and identified by 15% SDS-PAGE.

[0120] in, Figure 5 The images show the metal chelate chromatography purification chromatograms and electrophoretic identification results of the His-2Sumo-rhIL-2 protein in Example 3. Figure 5 (a) in the figure is the result of metal chelation chromatography. Figure 5 (b) SDS-PAGE purity results, (b) Lane 1: Protein Marker, Lane 2: rhIL-2 loaded sample, Lane 3: rhIL-2 flow-through sample, Lane 4: rhIL-2 eluted sample, Lane 5: rhIL-2 eluted sample.

[0121] Figure 6 The images show the chromatography patterns and electrophoretic identification of His-2Sumo-IL-2M metal chelate purification. Figure 6 (a) in the figure is the result of metal chelation chromatography. Figure 6 (b) SDS-PAGE purity characterization results, (b) Lane 1: Protein Marker, Lane 2: IL-2M loaded sample, Lane 3: IL-2M flow-through sample, Lane 4: IL-2M eluted sample, Lane 5: IL-2M eluted sample.

[0122] Figure 7 The images show the chromatographic patterns (a), (b), and electrophoretic identification (c) of the fusion protein purified by IL-2 and IL-2M affinity chromatography (flow-through elution mode) after ULP1 digestion. Figure 7(c) Lane 1: His-(Sumo)2-rhIL-2 before digestion; Lane 2: His-(Sumo)2-rhIL-2 after digestion; Lane 3: rhIL-2 flow-through 1; Lane 4: rhIL-2 flow-through 2; Lane 5: Column regeneration to elute His-(Sumo)2 sample; Lane 6: His-(Sumo)2-IL-2M before digestion; Lane 7: His-(Sumo)2-IL-2M after digestion; Lane 8: IL-2M flow-through 1; Lane 9: IL-2M flow-through 2; Lane 10: Column regeneration to elute His-(Sumo)2 sample. Example 4

[0123] Identification of the structure and activity of rhIL-2 and IL-2M proteins

[0124] Circular dichroism spectroscopy identification:

[0125] The secondary structures of rhIL-2 and rhIL-2M proteins were identified using circular dichroism spectroscopy. First, the protein sample buffer was replaced with 20 mM PB at pH 7.5, and the protein concentration was concentrated and adjusted to 0.3–0.5 mg / ml. A 0.1 cm thick sample cell was used, with a scanning wavelength interval of 1.0 nm, 5 scans, a scanning wavelength range of 190–260 nm, and a sample scanning speed of 1200 nm / min. The results showed that the secondary structures of rhIL-2 and rhIL-2M were consistent with the theoretical structures.

[0126] Fluorescence spectroscopy identification:

[0127] The secondary structures of rhIL-2 and rhIL-2M proteins were identified using a fluorescence spectrophotometer. First, the protein sample buffer was replaced with 20 mM PB, pH 7.5, and the protein concentration was concentrated and adjusted to approximately 0.1 mg / ml. The sample was placed in a quartz cuvette with a 1.0 cm optical path. The excitation wavelength was set to 280 nm, the emission wavelength to 280–450 nm, the scanning wavelength interval to 1.0 nm, and the sample scanning speed to 1000 nm / min. The results showed that the structures of rhIL-2 and rhIL-2M were compact.

[0128] Gel filtration analysis:

[0129] Gel filtration analysis was performed using the AKTA-PURE system. The gel filtration column was a Superdex™ 75Increase, and the buffer solution was 50 mM Na₂HPO₄ / NaH₂PO₄, 0.1 M Na₂SO₄, pH 7.4. The flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. The results showed that both IL-2 and IL-2M eluted outside the exclusion volume, indicating that IL-2 and IL-2M mainly existed in the form of soluble aggregates.

[0130] Cell viability assay:

[0131] Cell viability was assessed using CTLL-2 cells. CTLL-2 cells were cultured in RPMI-1640 medium containing 100 U / mL rhIL-2, 1.0 μg / mL ConA, and 10% fetal bovine serum. After three passages and stable cell growth, cells were harvested and the cell concentration was adjusted to 10⁻⁶ cells / mL using RPMI-1640 medium containing 1.0 μg / mL ConA and fetal bovine serum. 5 Cells were cultured at a concentration of 10 cells / ml in 96-well plates for 24 hours, followed by co-culturing with RPMI and fetal bovine serum containing different concentrations of IL-2 and IL-2 standards for 72 hours. Cell proliferation was then assessed using CCK-8 working solution. Results showed that the cell proliferation activity of IL-2 and IL-2 standards was similar.

[0132] in, Figure 8 The image shows a comparison of the circular dichroism spectral characterization of the prepared rhIL-2 and IL-2M. Figure 9 The image shows a comparison of the fluorescence spectral characterization of the prepared rhIL-2 and IL-2M. Figure 10 The results are obtained by high-performance gel filtration chromatography (HPLC) of rhIL-2 and IL-2M. Figure 11 The results are from an in vitro cell proliferation (CTLL-4) experiment using the prepared rhIL-2 and rhIL-2 standard proteins.

[0133] The method provided by this invention utilizes the tandem fusion of two or more Sumo sequences to achieve soluble expression of human IL-2 in an E. coli expression system. This avoids the formation of inclusion bodies in traditional E. coli IL-2 expression systems, prevents IL-2 and IL-2M from being expressed as unstructured and inactive inclusion bodies, and avoids the downstream IL-2 protein refolding process. Furthermore, it efficiently purifies rhIL-2 and IL-2M proteins from a complex bacterial lysate supernatant system through a simple chromatographic step. The method is characterized by its ease of operation, suitability for large-scale scaling, short cycle time, high purity, and stable process, achieving unexpected technical results.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for soluble expression and purification of recombinant human interleukin-2 or its mutants, characterized in that, Includes the following steps: Step 1: The gene sequence of human interleukin-2 or its mutant with the amino acid sequence shown in SEQ ID NO: 7 is spliced ​​to the 3' end of the gene sequence of two small ubiquitin-related modifiers to obtain the gene sequence of His-(Sumo)2-IL-2 or its mutant; wherein, the amino acid sequence of His-(Sumo)2 is shown in SEQ ID NO:

11. Step 2: Insert the gene sequence of His-(Sumo)2-IL-2 or its mutant obtained in Step 1 into the pET series expression plasmid vector, and then transform the plasmid vector into Origami B(DE3) competent cells; Step 3: Select dominant growth colonies by resistance plating, expand culture, and obtain the fusion protein His-(Sumo)2-IL-2 or its mutant by intervening in the promoter to transcribe and translate the target gene. Step 4: Lyse the bacterial cells, centrifuge to obtain the supernatant, and purify the supernatant to obtain high-purity recombinant His-(Sumo)2-IL-2 or its mutant fusion protein by the first metal chelate chromatography. After removing the His-(Sumo)2 fusion sequence by enzyme excision, the protein is then subjected to a second metal chelate chromatography to obtain recombinant human interleukin-2 or its mutant with the amino acid sequence shown in SEQ ID NO:

7.

2. The method for soluble expression and purification of recombinant human interleukin-2 or its mutants according to claim 1, characterized in that, In step 1, the amino acid sequence of human interleukin-2 or its mutant amino acid sequence are sequentially subjected to gene sequence conversion and E. coli host preference codon optimization to obtain the gene sequence of human interleukin-2 or its mutant amino acid sequence as shown in SEQ ID NO:

7.

3. The method for soluble expression and purification of recombinant human interleukin-2 or its mutants according to claim 2, characterized in that, The gene sequence of human interleukin-2 is shown in SEQ ID NO:

1.

4. The method for soluble expression and purification of recombinant human interleukin-2 or its mutants according to claim 1, characterized in that, In step 2, the plasmid vector is pET-32a plasmid.

5. The method for soluble expression and purification of recombinant human interleukin-2 or its mutants according to any one of claims 1-4, characterized in that, In step 4, the first metal chelation chromatography is in adsorption-elution mode; the second chelation chromatography is in flow-through mode.

6. The method for soluble expression and purification of recombinant human interleukin-2 or its mutants according to claim 5, characterized in that, In step 4, the solid-phase chromatography packing material used in the first and second metal chelation chromatography is a type of packing material that can be tandemly fused with three or more histidine residues for affinity binding and adsorption. The solid-phase chromatography microsphere packing material chelates divalent metal ions, including nickel ions, copper ions, zinc ions, and cobalt ions, by deriving chelating groups.

7. The method for soluble expression and purification of recombinant human interleukin-2 or its mutants according to claim 6, characterized in that, The specific steps in step 4 are as follows: Step 41: After expression, the bacterial cells are resuspended in resuspension buffer and then lysed to release intracellular proteins into extracellular resuspension buffer. The bacterial lysis supernatant is then centrifuged. Step 42: The supernatant obtained in step 41 is purified by nickel metal chelate chromatography. After the sample flows through the chromatography packing material, His-(Sumo)2-IL-2 or the mutant target protein is adsorbed on the chromatography packing material. After appropriate rinsing, the protein is finally eluted with imidazole elution buffer. The protein samples obtained in steps 43 and 42 were subjected to imidazole removal and buffer replacement. The buffer consisted of 50 mM PB and 100 mM arginine at pH 7.

0. Then, the His-(Sumo)2 fusion protein was digested with ULP1 enzyme to release free IL-2 or the mutant. The digestion time was 1 to 24 hours and the digestion temperature was 4 to 30°C. The mass ratio of ULP1 enzyme to protein was 1:10 to 10000. Step 44: The enzyme-digested sample obtained in step 43 is subjected to nickel metal chelate chromatography purification again. ULP1 enzyme, His-(Sumo)2 protein and His-(Sumo)2-IL-2 fusion protein that has not been effectively digested are adsorbed onto the chromatographic packing material. The target protein IL-2 or the mutant flows through the chromatographic column. Finally, the flow-through is collected, concentrated, and the medium is changed to obtain recombinant human interleukin-2 or the mutant with the amino acid sequence shown in SEQ ID NO: 7.

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