CN117683750A - Recombinant alpha-glucosidase and preparation method thereof - Google Patents
Recombinant alpha-glucosidase and preparation method thereof Download PDFInfo
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Abstract
The invention provides a recombinant alpha-glucosidase and a preparation method thereof, in particular to a recombinant alpha-glucosidase expressed by adopting genetically engineered escherichia coli, which realizes the soluble expression in an escherichia coli system, has high protein yield and higher catalytic activity. Therefore, the method has the advantages of short production period, easy purification of the expression product, low cost and the like, and can realize the industrialized production of the recombinant alpha-glucosidase.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant alpha-glucosidase and a preparation method thereof.
Background
Alpha-glucosidase, also known as alpha-D-glucosidase or glucosyltransferase, belongs to the class of amylohydrolases. It hydrolyzes the alpha-1, 4 glycosidic bond at the non-reducing end of the maltooligosaccharide molecule, and cleavage of the glycosidic bond occurs between the anomeric carbon of the glycosyl and the glycosidic oxygen atom. And can transfer and combine one glucose residue which is released by hydrolysis to another glucose molecule or maltose, isomaltotriose and the like. Meanwhile, the oligosaccharide has transglycosylation, and can convert alpha-1, 4-glycosidic bond in the oligosaccharide into alpha-1, 6-glycosidic bond, so that the isomaltooligosaccharide or sugar ester, glycopeptide and the like can be obtained through the catalysis of enzyme without adopting a chemical approach.
Alpha-glucosidase is a key enzyme for industrial production of isomaltose hypgather such as isomaltose, isomaltotriose and the like. The stevioside derivative synthesized by utilizing the transglycosylation has high sweetness and can be used as an excellent sweetener in the food industry. The alpha-glucosidase forms isomaltose, panose, isomaltotriose and other isomaltose oligomers during the beer brewing process, so that the taste of the beer can be improved. Alpha-glucosidase has been gaining attention for many years because of its important role in starch processing. Compared with normal temperature alpha-glucosidase, the thermophilic enzyme has the advantages of good heat stability, high activity, wide application range, low purification cost and the like, and is more suitable for industrial application. A great deal of research shows that the application of the alpha-glucosidase in the industrial field can bring good economic and social benefits.
Therefore, those skilled in the art have been working on the engineering and recombinant expression of the alpha-glucosidase in order to prepare the alpha-glucosidase suitable for industrial production at low cost and maintaining high reaction efficiency.
Disclosure of Invention
The invention aims to provide a recombinant alpha-glucosidase and a preparation method thereof.
In a first aspect of the invention, a recombinant α -glucosidase is provided, the amino acid sequence of which is shown in SEQ ID No.1, or the amino acid sequence of which has at least 80% homology to SEQ ID No. 1; more preferably, it has a homology of at least 90%; most preferably, having at least 95% homology; such as having at least 96%, 97%, 98%, 99% homology.
In a second aspect of the invention there is provided a polynucleotide molecule encoding a recombinant α -glucosidase according to the first aspect of the invention.
In another preferred embodiment, the polynucleotide molecule sequence is shown in SEQ ID NO. 4.
In a third aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.
In a fourth aspect of the invention there is provided a host cell comprising a vector or chromosome according to the first aspect of the invention incorporating a nucleic acid molecule according to the second aspect of the invention.
In another preferred embodiment, the host cell is a prokaryotic cell, or a eukaryotic cell.
In another preferred embodiment, the prokaryotic cell is E.coli.
In a fifth aspect of the invention, there is provided a method for preparing a recombinant α -glucosidase according to the first aspect of the invention, comprising the steps of:
(i) Culturing the host cell of the fourth aspect of the invention under suitable conditions to express said recombinant α -glucosidase; and
(ii) Isolating said recombinant α -glucosidase.
In another preferred embodiment, the temperature at which the host cells are cultured in step (i) is from 20℃to 40 ℃; preferably from 25℃to 37℃such as 37 ℃.
In another preferred embodiment, said host cell in step (i) is an E.coli cell.
In a sixth aspect of the invention, a kit is provided, said kit comprising the recombinant α -glucosidase according to the first aspect of the invention.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the results of recombinant alpha-glucosidase expression test;
FIG. 2 is a graph showing the standard of enzyme activity detection.
Detailed Description
Through extensive and intensive research, the inventor adopts genetically engineered escherichia coli to express recombinant alpha-glucosidase, and the expressed recombinant alpha-glucosidase realizes soluble expression in an escherichia coli system, has high protein yield and higher catalytic activity. Therefore, the method has the advantages of short production period, easy purification of the expression product, low cost and the like, and can realize the industrialized production of the recombinant alpha-glucosidase. On this basis, the present invention has been completed.
Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
In a preferred embodiment of the invention, the amino acid sequence of the recombinant α -glucosidase according to the invention (SEQ ID NO. 1) is as follows:
MKKTWWKEGVAYQIYPRSFMDANGDGIGDLRGI IEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKIILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHLFDVKQPDLNWENSEVRQALYDMINWWLDKGIDGFRIDAISHIKKKPGLPDLPNPKGLKYVPSFAAHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFHMIFQFEHLGLWKRKADGSIDVRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDREYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVQFSDIRDYRDVAALRLYELERANGRTHEEVMKIIWKTGRDNSRTPMQWSDAPNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGAYDLLLENHPSIYAYTRTLGRDRALI IVNVSDRPSLYRYDGFRLQSSDLALSNYPVREHKNATRFKLKPYEARVYIWKE
in a preferred embodiment of the invention, the gene sequence of the recombinant α -glucosidase is as follows (optimized for synonymous codon preference for E.coli, SEQ ID NO. 4):
ATGAAGAAGACTTGGTGGAAAGAAGGCGTTGCGTATCAGATTTACCCTCGTTCTTTCATGGATGCTAACGGCGACGGTATCGGCGACCTGCGTGGCATCATCGAGAAACTGGACTACCTGGTAGAACTGGGTGTGGATATCGTGTGGATTTGCCCGATCTACCGTAGCCCGAACGCAGACAACGGCTATGACATTTCCGACTACTACGCAATCATGGATGAGTTTGGTACCATGGACGATTTCGACGAACTGCTGGCGCAGGCACACCGTCGTGGTCTGAAAATTATTCTGGATCTGGTGATCAACCACACCAGCGATGAACACCCGTGGTTCATCGAAAGCCGTTCTTCCCGTGACAACCCTAAACGCGATTGGTACATCTGGCGTGATGGTAAGGACGGTCGTGAACCGAACAACTGGGAATCCATCTTCGGCGGTTCCGCATGGCAGTATGACGAACGCACCGGCCAGTATTATCTGCACCTGTTTGACGTTAAACAGCCGGATCTGAACTGGGAAAACTCCGAAGTTCGCCAAGCACTGTATGATATGATCAACTGGTGGCTGGACAAAGGCATCGACGGTTTCCGCATCGACGCGATCTCCCACATCAAAAAGAAACCGGGTCTGCCTGACCTGCCGAACCCGAAAGGCCTGAAATACGTCCCGAGCTTCGCAGCTCACATGAACCAACCGGGCATTATGGAATATCTGCGTGAACTGAAAGAGCAGACCTTTGCCCGTTACGATATCATGACCGTGGGTGAAGCCAACGGCGTGACGGTTGACGAAGCGGAACAGTGGGTAGGTGAAGAAAACGGTGTCTTCCACATGATTTTCCAGTTTGAACACCTGGGTCTGTGGAAACGCAAGGCTGACGGTTCCATTGACGTTCGTCGTCTGAAACGTACCCTGACTAAATGGCAGAAAGGTCTGGAAAACCGCGGTTGGAATGCCCTGTTCCTGGAAAACCATGATCTGCCGCGTAGCGTTAGCACCTGGGGCAATGATCGTGAATATTGGGCCGAAAGCGCCAAAGCGCTGGGCGCCCTGTACTTCTTCATGCAGGGCACCCCATTCATCTACCAGGGTCAGGAAATCGGCATGACGAACGTGCAATTCTCTGATATTCGTGACTATCGTGACGTTGCCGCGCTGCGTCTGTACGAACTGGAACGTGCTAACGGTCGTACTCATGAAGAAGTGATGAAAATCATCTGGAAAACCGGCCGCGACAACTCCCGTACGCCGATGCAGTGGTCCGACGCACCGAACGCCGGCTTCACGACCGGCACTCCGTGGATCAAAGTTAACGAAAACTACCGCACGATCAACGTTGAGGCAGAACGTCGTGATCCGAATTCTGTTTGGTCTTTCTATCGTCAGATGATTCAGCTGCGCAAAGCGAACGAACTGTTTGTTTACGGTGCATACGACCTGCTGCTGGAAAACCATCCGTCTATCTACGCCTACACCCGTACCCTGGGTCGCGACCGTGCTCTGATCATCGTTAACGTCTCCGACCGTCCGTCCCTGTACCGTTACGACGGCTTCCGCCTGCAGTCCTCTGATCTGGCGCTGAGCAACTATCCGGTACGCGAGCACAAAAACGCAACTCGTTTCAAACTGAAACCGTATGAGGCTCGCGTTTATATCTGGAAGGAA
the recombinant enzyme gene sequences of the present invention may be obtained by conventional methods, such as total artificial synthesis or PCR synthesis, which can be used by those of ordinary skill in the art. One preferred synthesis method is an asymmetric PCR method. The asymmetric PCR method is to amplify a large amount of single-stranded DNA (ssDNA) by PCR using a pair of primers in unequal amounts. The pair of primers is referred to as non-limiting primer and limiting primer, respectively, in a ratio of typically 50-100:1. During the first 10-15 cycles of the PCR reaction, the amplified product is mainly double stranded DNA, but when the restriction primer (low concentration primer) is consumed, the non-restriction primer (high concentration primer) directed PCR will produce a large amount of single stranded DNA. Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
The mutant enzymes of the invention may be expressed or produced by conventional recombinant DNA techniques comprising the steps of:
(1) Transforming or transducing a suitable host cell with a polynucleotide encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) Culturing the host cell in a suitable medium;
(3) And separating and purifying the target protein from the culture medium or the cells to obtain the target enzyme.
Methods well known to those skilled in the art can be used to construct expression vectors comprising the coding DNA sequences for the enzymes of the invention and appropriate transcriptional/translational control signals, preferably commercially available vectors: pET28. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
The recombinant vector comprises in the 5 'to 3' direction: a promoter, a gene of interest and a terminator. If desired, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polynucleotide acidification signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; selection markers (antibiotic resistance genes, fluorescent proteins, etc.); an enhancer; or an operator.
Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid or vector may be used as long as it is capable of replication and stability in a host.
The person skilled in the art can construct vectors containing the promoter and/or the gene sequence of interest of the present invention by means of well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
The expression vectors of the invention may be used to transform an appropriate host cell to allow the host to transcribe the RNA of interest or to express the protein of interest. The host cell may be a prokaryotic cell such as E.coli, corynebacterium glutamicum, brevibacterium flavum, streptomyces, agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., E.coli), caCl may be used 2 The treatment can also be carried out by electroporation. When (when)The host is eukaryotic, and the following DNA transfection method can be selected: calcium phosphate co-precipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may also be transformed by Agrobacterium or gene gun, such as leaf disc method, embryo transformation method, flower bud soaking method, etc. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain transgenic plants.
The term "operably linked" refers to the attachment of a gene of interest to be expressed by transcription to its control sequences in a manner conventional in the art.
Culturing engineering bacteria and fermenting production of target protein
After obtaining the engineered cells, the engineered cells may be cultured under appropriate conditions to express the protein encoded by the gene sequence of the present invention. The medium used in the culture may be selected from various conventional media according to the host cell, and the culture is performed under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
In the present invention, conventional fermentation conditions may be employed. Representative conditions include (but are not limited to):
(a) In terms of temperature, the fermentation and induction temperatures of the enzymes are maintained at 25-37 ℃;
(b) The pH value in the induction period is controlled to be 3-9;
(c) In the case of Dissolved Oxygen (DO), the DO is controlled to be 10-90%, and the maintenance of dissolved oxygen can be solved by the introduction of oxygen/air mixed gas;
(d) For the feeding, the type of the feeding preferably comprises carbon sources such as glycerol, methanol, glucose and the like, and the feeding can be carried out independently or by mixing;
(e) As for the induction period IPTG concentration, conventional induction concentrations can be used in the present invention, and usually the IPTG concentration is controlled to 0.1-1.5mM;
(f) The induction time is not particularly limited, and is usually 2 to 20 hours, preferably 5 to 15 hours.
The target protein of the invention exists in E.coli cells, host cells are collected by a centrifuge, and then the host cells are broken up by high pressure, mechanical force, enzymatic hydrolysis cell cover or other cell disruption methods to release recombinant protein, preferably a high pressure method. The host cell lysate can be purified primarily by flocculation, salting out, ultrafiltration and other methods, and then subjected to chromatography, ultrafiltration and other purification methods, or can be directly subjected to chromatography purification.
The chromatographic techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. Common chromatographic methods include:
1. anion exchange chromatography:
anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is high, which affects the binding to the ion exchange medium, the salt concentration is reduced before ion exchange chromatography is performed. The sample can be replaced by dilution, ultrafiltration, dialysis, gel filtration chromatography and other means until the sample is similar to the corresponding ion exchange column equilibrium liquid system, and then the sample is loaded to perform gradient elution of salt concentration or pH.
2. Hydrophobic chromatography:
hydrophobic chromatography media include (but are not limited to): phenyl-Sepharose, butyl-Sepharose, octyle-Sepharose. Sample by adding NaCl, (NH) 4 ) 2 SO 4 And the salt concentration is increased in an equal mode, then the sample is loaded, and the sample is eluted by a method of reducing the salt concentration. The hetero proteins with a large difference in hydrophobicity were removed by hydrophobic chromatography.
3. Gel filtration chromatography
Hydrophobic chromatography media include (but are not limited to): sephacryl, superdex, sephadex. The buffer system is replaced by gel filtration chromatography or further purified.
4. Affinity chromatography
Affinity chromatography media include (but are not limited to): hiTrap TM HeparinHPColumns。
5. Membrane filtration
The ultrafiltration medium comprises: organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. The purposes of purification and concentration can be achieved by membrane filtration.
The invention has the main advantages that:
(1) The recombinant alpha-glucosidase can realize a large amount of soluble expression in an escherichia coli expression system, is easy to purify and high in yield, and keeps higher specific enzyme activity.
(2) The invention adopts genetically engineered escherichia coli recombinant fusion to express alpha-glucosidase, and provides a method for industrially producing the alpha-glucosidase. The method has the advantages of simple purification steps, high activity of the produced enzyme, high protein expression and low production cost.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
EXAMPLE 1 expression and purification of recombinant proteins
The gene of alpha-glucosidase from wild thermophilic bacillus (WP_ 096225765.1), aspergillus oryzae and Thermus maritimus is used as reference, the sequence is optimized by the preference of synonymous codons of escherichia coli, the connection carrier is pET-28a (+), and the expression is carried out by escherichia coli BL21 (DE 3) strain synthesized by Suzhou Jin Weizhi biotechnology Co.
The result shows that alpha-glucosidase from Thermomyces maritimus can be expressed in the supernatant in E.coli expression strain; the thermophilic bacillus and aspergillus oryzae sources are inclusion body expression. The bacterial lysate qualitatively measures that only the crude enzyme solution derived from thermophilic bacillus is active, so that the alpha-glucosidase derived from thermophilic bacillus is selected for subsequent experiments.
Based on a wild thermophilic bacillus-derived alpha-glucosidase protein sequence (WP_ 096225765.1), the structure of the recombinant alpha-glucosidase protein is optimized by using computer molecular simulation so as to avoid inclusion body expression in escherichia coli, and then codon optimization is performed on the recombinant alpha-glucosidase protein subjected to amino acid optimization so as to improve the protein expression quantity and enzyme activity of the recombinant alpha-glucosidase.
The alpha-glucosidase protein sequence after structure optimization is as follows (SEQ ID NO. 1): MKKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKIILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHLFDVKQPDLNWENSEVRQALYDMINWWLDKGIDGFRIDAISHIKKKPGLPDLPNPKGLKYVPSFAAHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFHMIFQFEHLGLWKRKADGSIDVRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDREYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVQFSDIRDYRDVAALRLYELERANGRTHEEVMKIIWKTGRDNSRTPMQWSDAPNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGAYDLLLENHPSIYAYTRTLGRDRALIIVNVSDRPSLYRYDGFRLQSSDLALSNYPVREHKNATRFKLKPYEARVYIWKE
Gene sequence 1 (SEQ ID No. 2) of the codon-optimized recombinant α -glucosidase: ATGAAAAAAACCTGGTGGAAAGAAGGTGTTGCTTACCAGATCTACCCGCGTTCTTTCATGGACGCTAACGGTGACGGTATCGGTGACCTGCGTGGTATCATCGAAAAACTGGACTACCTGGTTGAACTGGGTGTTGACATCGTTTGGATCTGCCCGATCTACCGTTCTCCGAACGCTGACAACGGTTACGACATCTCTGACTACTACGCTATCATGGACGAATTCGGTACCATGGACGACTTCGACGAACTGCTGGCTCAGGCTCACCGTCGTGGTCTGAAAATCATCCTGGACCTGGTTATCAACCACACCTCTGACGAACACCCGTGGTTCATCGAATCTCGTTCTTCTCGTGACAACCCGAAACGTGACTGGTACATCTGGCGTGACGGTAAAGACGGTCGTGAACCGAACAACTGGGAATCTATCTTCGGTGGTTCTGCTTGGCAGTACGACGAACGTACCGGTCAGTACTACCTGCACCTGTTCGACGTTAAACAGCCGGACCTGAACTGGGAAAACTCTGAAGTTCGTCAGGCTCTGTACGACATGATCAACTGGTGGCTGGACAAAGGTATCGACGGTTTCCGTATCGACGCTATCTCTCACATCAAAAAAAAACCGGGTCTGCCGGACCTGCCGAACCCGAAAGGTCTGAAATACGTTCCGTCTTTCGCTGCTCACATGAACCAGCCGGGTATCATGGAATACCTGCGTGAACTGAAAGAACAGACCTTCGCTCGTTACGACATCATGACCGTTGGTGAAGCTAACGGTGTTACCGTTGACGAAGCTGAACAGTGGGTTGGTGAAGAAAACGGTGTTTTCCACATGATCTTCCAGTTCGAACACCTGGGTCTGTGGAAACGTAAAGCTGACGGTTCTATCGACGTTCGTCGTCTGAAACGTACCCTGACCAAATGGCAGAAAGGTCTGGAAAACCGTGGTTGGAACGCTCTGTTCCTGGAAAACCACGACCTGCCACGTTCTGTAAGCACCTGGGGTAACGACCGTGAATACTGGGCTGAATCTGCTAAAGCTCTGGGTGCTCTGTACTTCTTCATGCAGGGTACCCCGTTCATCTACCAGGGTCAGGAAATCGGTATGACCAACGTTCAGTTCTCTGACATCCGTGACTACCGTGACGTTGCTGCTCTGCGTCTGTACGAACTGGAACGTGCTAACGGTCGTACCCACGAAGAAGTTATGAAAATCATCTGGAAAACCGGTCGTGACAACTCTCGTACCCCGATGCAGTGGTCTGACGCTCCAAATGCTGGCTTCACCACCGGTACTCCGTGGATCAAAGTTAACGAAAACTACCGTACCATCAACGTTGAAGCTGAACGTCGTGACCCCAATTCTGTTTGGAGCTTCTACCGTCAGATGATCCAGCTGCGTAAAGCTAATGAACTCTTCGTCTACGGCGCTTACGACCTGCTGCTGGAAAACCACCCGTCTATCTACGCTTACACCCGTACCCTGGGTCGTGACCGTGCTCTGATCATCGTTAACGTTTCTGACCGTCCGTCTCTGTACCGTTACGATGGCTTCCGTCTGCAGTCGTCTGACCTGGCTCTGTCTAACTACCCGGTTCGTGAACACAAAAACGCTACCCGTTTCAAACTGAAACCGTACGAAGCTCGTGTTTACATCTGGAAAGAA
Gene sequence 2 (SEQ ID No. 3) of the codon-optimized recombinant α -glucosidase: ATGAAAAAGACGTGGTGGAAGGAAGGTGTTGCGTATCAAATTTACCCACGCAGTTTCATGGACGCGAATGGCGACGGTATCGGTGATCTTCGCGGAATTATCGAAAAGCTGGACTACCTGGTGGAGCTTGGAGTTGATATTGTGTGGATTTGCCCCATCTATCGTTCTCCCAACGCAGATAACGGTTACGATATTTCAGACTATTATGCAATTATGGACGAGTTCGGTACGATGGACGACTTTGACGAACTGTTAGCGCAAGCCCACCGTCGCGGGTTAAAAATCATCTTGGATTTAGTAATTAACCACACCAGTGATGAACACCCTTGGTTTATCGAGAGCCGCTCATCCCGTGACAACCCCAAGCGCGATTGGTACATCTGGCGCGACGGAAAGGACGGGCGCGAACCGAACAATTGGGAAAGTATCTTTGGTGGCTCGGCCTGGCAGTACGATGAACGCACCGGGCAATATTACCTGCATCTTTTCGACGTAAAACAGCCAGACTTGAACTGGGAGAACTCGGAAGTACGCCAGGCATTATACGACATGATCAACTGGTGGTTGGATAAGGGAATCGATGGTTTTCGCATTGATGCTATCAGTCATATCAAGAAAAAACCGGGGCTTCCGGATTTACCGAATCCAAAGGGTCTTAAATACGTACCCTCTTTCGCTGCTCATATGAACCAGCCAGGGATTATGGAGTATCTGCGTGAGCTGAAAGAGCAAACATTTGCGCGTTACGACATTATGACCGTAGGCGAGGCAAATGGAGTCACCGTAGACGAGGCTGAACAATGGGTTGGCGAGGAGAATGGCGTGTTTCATATGATCTTCCAATTTGAGCATTTGGGTTTATGGAAACGTAAAGCCGATGGCTCAATTGACGTCCGTCGTCTTAAACGCACTCTGACTAAATGGCAAAAGGGTTTAGAGAACCGTGGGTGGAACGCGTTGTTCTTGGAAAACCATGATCTGCCACGTTCTGTCTCTACGTGGGGGAATGATCGCGAATATTGGGCCGAATCGGCAAAGGCATTAGGCGCATTATATTTTTTTATGCAGGGCACCCCTTTTATTTACCAGGGGCAGGAGATTGGAATGACGAATGTCCAGTTTTCAGACATTCGTGACTATCGTGACGTTGCCGCCTTGCGCCTTTACGAGTTGGAGCGTGCGAATGGTCGCACGCATGAAGAAGTGATGAAGATCATCTGGAAAACAGGGCGCGATAACTCCCGTACTCCGATGCAATGGAGCGATGCACCCAATGCTGGGTTTACGACAGGTACACCTTGGATTAAAGTCAATGAGAACTACCGCACCATCAATGTTGAGGCTGAGCGCCGCGATCCCAATTCCGTTTGGTCATTTTACCGCCAAATGATTCAACTGCGTAAGGCTAACGAATTGTTTGTGTACGGCGCGTACGATTTACTGCTTGAGAACCACCCCTCCATCTACGCATATACGCGCACTTTAGGGCGCGATCGCGCCTTGATTATTGTTAACGTCTCCGACCGTCCCTCGTTATATCGTTACGATGGTTTCCGCCTTCAGAGCTCCGATCTGGCACTGAGTAATTATCCGGTGCGTGAGCACAAAAATGCGACGCGCTTCAAACTTAAGCCATACGAAGCACGCGTTTACATTTGGAAAGAA
Gene sequence 3 (SEQ ID No. 4) of the codon-optimized recombinant α -glucosidase: ATGAAGAAGACTTGGTGGAAAGAAGGCGTTGCGTATCAGATTTACCCTCGTTCTTTCATGGATGCTAACGGCGACGGTATCGGCGACCTGCGTGGCATCATCGAGAAACTGGACTACCTGGTAGAACTGGGTGTGGATATCGTGTGGATTTGCCCGATCTACCGTAGCCCGAACGCAGACAACGGCTATGACATTTCCGACTACTACGCAATCATGGATGAGTTTGGTACCATGGACGATTTCGACGAACTGCTGGCGCAGGCACACCGTCGTGGTCTGAAAATTATTCTGGATCTGGTGATCAACCACACCAGCGATGAACACCCGTGGTTCATCGAAAGCCGTTCTTCCCGTGACAACCCTAAACGCGATTGGTACATCTGGCGTGATGGTAAGGACGGTCGTGAACCGAACAACTGGGAATCCATCTTCGGCGGTTCCGCATGGCAGTATGACGAACGCACCGGCCAGTATTATCTGCACCTGTTTGACGTTAAACAGCCGGATCTGAACTGGGAAAACTCCGAAGTTCGCCAAGCACTGTATGATATGATCAACTGGTGGCTGGACAAAGGCATCGACGGTTTCCGCATCGACGCGATCTCCCACATCAAAAAGAAACCGGGTCTGCCTGACCTGCCGAACCCGAAAGGCCTGAAATACGTCCCGAGCTTCGCAGCTCACATGAACCAACCGGGCATTATGGAATATCTGCGTGAACTGAAAGAGCAGACCTTTGCCCGTTACGATATCATGACCGTGGGTGAAGCCAACGGCGTGACGGTTGACGAAGCGGAACAGTGGGTAGGTGAAGAAAACGGTGTCTTCCACATGATTTTCCAGTTTGAACACCTGGGTCTGTGGAAACGCAAGGCTGACGGTTCCATTGACGTTCGTCGTCTGAAACGTACCCTGACTAAATGGCAGAAAGGTCTGGAAAACCGCGGTTGGAATGCCCTGTTCCTGGAAAACCATGATCTGCCGCGTAGCGTTAGCACCTGGGGCAATGATCGTGAATATTGGGCCGAAAGCGCCAAAGCGCTGGGCGCCCTGTACTTCTTCATGCAGGGCACCCCATTCATCTACCAGGGTCAGGAAATCGGCATGACGAACGTGCAATTCTCTGATATTCGTGACTATCGTGACGTTGCCGCGCTGCGTCTGTACGAACTGGAACGTGCTAACGGTCGTACTCATGAAGAAGTGATGAAAATCATCTGGAAAACCGGCCGCGACAACTCCCGTACGCCGATGCAGTGGTCCGACGCACCGAACGCCGGCTTCACGACCGGCACTCCGTGGATCAAAGTTAACGAAAACTACCGCACGATCAACGTTGAGGCAGAACGTCGTGATCCGAATTCTGTTTGGTCTTTCTATCGTCAGATGATTCAGCTGCGCAAAGCGAACGAACTGTTTGTTTACGGTGCATACGACCTGCTGCTGGAAAACCATCCGTCTATCTACGCCTACACCCGTACCCTGGGTCGCGACCGTGCTCTGATCATCGTTAACGTCTCCGACCGTCCGTCCCTGTACCGTTACGACGGCTTCCGCCTGCAGTCCTCTGATCTGGCGCTGAGCAACTATCCGGTACGCGAGCACAAAAACGCAACTCGTTTCAAACTGAAACCGTATGAGGCTCGCGTTTATATCTGGAAGGAA
The sequence optimized by the synonymous codon preference of the escherichia coli is synthesized by the biological technology limited company of Suzhou Jin Weizhi, and the connecting carrier is pET-28a (+).
Taking 1 mu L of expression plasmid, adding the expression plasmid into 30 mu L of escherichia coli competent BL21 (DE 3) under ice bath condition, standing in ice bath for 30min, standing in water bath at 42 ℃ for 45s, standing on ice immediately for 2min, adding 400 mu L of SOC culture medium without antibiotics, and culturing at 37 ℃ and 230rpm for 45min in a shaking way. mu.L of the bacterial liquid was uniformly spread on LB plates containing 100. Mu.g/mL of kana resistance, and incubated overnight at 37 ℃.
The monoclonal is selected, sterile operation is respectively inoculated into LB culture medium containing 100 mug/mL kana resistance, and the culture is carried out repeatedly, 220rpm shaking culture is carried out at 37 ℃ until OD600 is between 0.6 and 0.8, IPTG is induced, and then the culture is carried out at 37 ℃ shaking culture overnight. SDS-PAGE was performed by sampling ultrasonication to identify expression of the recombinant protein.
FIG. 1 shows the results of recombinant proteins expressed at different codons, lane 1 being a molecular weight marker, lane 2 being wild-type α -glucosidase, lane 3 being a codon-optimized gene sequence 1, lane 4 being a codon-optimized gene sequence 2, and lane 5 being a codon-optimized gene sequence 3.
Protein purification of the fermentation product:
dialysis Buffer:10 XPBS diluted to 1 XPBS pH7.4
About 4g of cells were weighed, and 20ml of Lysis Buffer was added thereto to disperse the cells on ice using a disperser. Ultrasonic disruption of cells: the phi 10 probe has 10 percent of power, works for 5.5 seconds, stops for 9.9 seconds and is subjected to ultrasonic crushing for 30 minutes. Centrifugation was carried out at 20000rpm at 4℃for 30min, and the supernatant was collected and filtered through a 0.22 μm membrane.
Purification was performed with 1ml Ni-NTA at a flow rate of 0.5ml/min, UV washed and conducted to baseline using 20ml Lysis Buffer. The elution procedure included: step 1:0% B,10CV,2ml/min; step2, 0-60% of B,20CV,2ml/min; step 3:100% B,10CV,0ml/min. The electrophoresis result shows that the peak obtained by purification is the target protein peak, and the protein purity is higher.
And (3) selecting 21ml of purified samples according to SDS results, putting the purified samples into a 10KD dialysis bag, putting the 10KD dialysis bag into 1300ml of dialysate for overnight dialysis, collecting the dialyzed samples in the morning the next day, uniformly mixing the samples, and storing the samples in a-80 refrigerator with the volume of 19 ml.
The protein concentration of the codon optimized gene sequence 1 is measured to be 0.38mg/ml by using a BCA method, the yield is 7.22mg, and the yield is 1.8mg/g bacteria; the protein concentration of the codon optimized gene sequence 2 is 0.16mg/ml, the yield is 3.04mg, and the yield is 0.76mg/g bacteria; the protein concentration of the codon optimized gene sequence 3 is 0.84mg/ml, the yield is 15.96mg, and the yield is 3.99mg/g bacteria. The protein expression level of the codon optimized gene sequence 3 is obviously superior to other codons.
EXAMPLE 2 determination of alpha-glucosidase Activity
1) Experimental procedure
(1) Configuration of positive enzymes: 1KU positive enzyme was dissolved in 100. Mu.L of PBS pH7.4 buffer to prepare 10U/. Mu.L of enzyme solution.
(2) The enzyme label instrument is preheated for 30min
(3) The reaction system is as follows:
detection of alpha-glucosidase (alpha-GC) Activity kit (Soy Bao, BC 2550)
(4) Program setting of the enzyme label instrument: the blank tube and the measuring tube draw 100 mu L of reaction liquid into the ELISA plate, and OD value is read at 400 nm.
(5) Calculating the OD difference A2-A1 between the sample and the blank
The results of the α -glucosidase standard curve are shown in FIG. 2.
The absorbance of each enzyme was measured as described above, and the enzyme activity was calculated as follows:
| group of | Enzyme activity (U/mL) | Specific activity (U/mg) |
| Codon 1 | 2.7 | 7.1 |
| Codon 2 | 2.1 | 13.1 |
| Codon 3 | 11.3 | 13.5 |
The result shows that the specific activity of the recombinant alpha-glucosidase expressed by the codon optimization gene sequence 1 is lower, and the specific activity of the recombinant alpha-glucosidase expressed by the codon optimization gene sequence 3 is obviously superior to other codons.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. A recombinant α -glucosidase characterized in that the amino acid sequence of the recombinant α -glucosidase is shown in SEQ ID No.1, or the amino acid sequence of the recombinant α -glucosidase has at least 80% homology to SEQ ID No. 1.
2. A polynucleotide molecule encoding the recombinant α -glucosidase of claim 1.
3. The polynucleotide molecule of claim 2, wherein the sequence of said polynucleotide molecule is set forth in SEQ ID No. 4.
4. A vector comprising the polynucleotide molecule of claim 2.
5. A host cell comprising the vector of claim 4 or a chromosome incorporating the polynucleotide molecule of claim 2.
6. The host cell of claim 5, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
7. A method for preparing the recombinant α -glucosidase of claim 1, comprising the steps of:
(i) Culturing the host cell of claim 5 under suitable conditions to express said recombinant α -glucosidase; and
(ii) Isolating said recombinant α -glucosidase.
8. The method of claim 7, wherein the temperature at which the host cells are cultured in step (i) is from 20 ℃ to 40 ℃.
9. The method of claim 7, wherein the host cell in step (i) is an e.
10. The method of claim 8, wherein the temperature at which the host cells are cultured in step (i) is from 25 ℃ to 37 ℃.
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