CN116042588A

CN116042588A - Preparation method and application of recombinant creatine enzyme

Info

Publication number: CN116042588A
Application number: CN202211391019.4A
Authority: CN
Inventors: 朱伟伟; 蒋析文; 张伟; 刘春奇; 陈园园; 林海辉
Original assignee: Guangzhou Da'an Gene Co ltd
Current assignee: Guangzhou Da'an Gene Co ltd
Priority date: 2022-11-08
Filing date: 2022-11-08
Publication date: 2023-05-02

Abstract

The application discloses a preparation method and application of recombinant creatine enzyme. In the application, a creatine enzyme preparation method based on a genetic engineering technology is developed, and polynucleotides encoding creatine enzyme optimized by synonymous codon preference are introduced into a vector to successfully construct a prokaryotic expression plasmid, so that the expression quantity of a target protein is improved; the recombinant creatine enzyme has high activity after the optimization of culture conditions such as culture medium, culture temperature and the like and the screening of combined strain sources, so that the yield of the soluble target protein is further improved.

Description

Preparation method and application of recombinant creatine enzyme

Technical Field

The invention relates to the field of genetic engineering, in particular to a preparation method and application of recombinant creatine enzyme.

Background

The creatinase is derived from microorganisms and consists of a single subunit or 2 identical subunits, the molecular weight of the subunits is in the range of 45-51kDa, for example, the molecular weight of the creatinase of P.putida var. Naraensis is 94kD, and consists of two identical subunits of 47kD, each subunit has a sulfhydryl group, and the groups are closely related to the activity of the creatinase; the creatine enzyme of Alcaligenes is single subunit enzyme, the molecular weight is 51kD, and the molecular biology method is used for cloning and expressing creatine hydrolase genes, so that the obtained recombinases are single subunit enzymes, and the molecular weight is 42-50 kD; such as Flavobacterium sp.U-188, having a molecular weight of 42.7kD.

Creatinine is an important metabolite in humans, and the creatinine content in the blood is typically maintained at 35-150 μmol/L, with excess creatinine being expelled to the outside through the renal system. Once the kidney function of the human body is problematic, creatinine cannot be discharged along with urine, the creatinine content in blood is increased, and the kidney function can be reflected by measuring the creatinine content in the blood and the urine.

Creatinine assays are mainly chemical and enzymatic, both of which are based on colorimetric methods. The chemical method mainly originates from the Jeffe reaction method, has the advantages of low cost, simple operation and easy development of basic units, and is still used in a plurality of basic hospitals at present, but has the obvious defect of being easy to be interfered by nonspecific substances in samples, namely the so-called pseudocreatinine. The enzyme method overcomes the defects, and has the advantages which are incomparable with chemical methods in both sensitivity and specificity, but has the defects of the enzyme method for measuring creatinine. The clinical creatinine enzymology detection mainly uses an imported original kit or a kit assembled by imported tool enzymes of domestic biotechnology company, and the cost is much higher than that of a chemical method. The enzymatic method mainly comprises 3 enzymatic reactions, wherein 3 key enzymes are creatininase, creatinase and sarcosine oxidase respectively. CRE acts as a key enzyme in enzymatic assays for creatinine, which can be broken down into sarcosine and urea.

In nature, a wide variety of strains can produce CRE under induction conditions, including: bacillus (Bacillus), flavobacterium (Flavobacterium), pseudomonas (Pseudomonas), arthrobacter (Alcaligenes), paracoccus (Paracoccus), clostridium (Clostridium), etc. Accordingly, there are also studies related to purification, property analysis, evaluation, etc. of CRE produced against wild bacteria. The international method for producing CRE has been found to give enzymes having good enzymatic properties but low yields and poor enzymatic activity. CRE is purified in Alcaligenes, such as Matsuda Y, etc., with very low wild type yields and only 0.4U/mL enzyme activity. At present, creatine enzyme cannot be produced autonomously in China. Therefore, there is still a need to develop a method for efficiently producing high-activity creatinase to meet the demand of creatinine assay.

Disclosure of Invention

The invention aims to provide a preparation method of recombinant creatine enzyme.

It is another object of the present invention to provide a polynucleotide sequence encoding a creatinase.

It is another object of the present invention to provide a vector adapted to a polynucleotide sequence encoding a creatinase.

It is another object of the present invention to provide a kit comprising a polynucleotide sequence encoding a creatinase.

To solve the above technical problem, according to a first aspect of the present invention, there is provided a polynucleotide encoding creatinase, the polynucleotide being codon optimized and the polynucleotide being selected from any one of the following:

(i) A polynucleotide having a sequence as shown in SEQ ID NO.2 or SEQ ID NO. 4;

(ii) Polynucleotides having greater than 95% homology to the sequences as set forth in SEQ ID No.2 or SEQ ID No. 4; and

(iii) Having a polynucleotide complementary to the polynucleotide sequence set forth in (i) or (ii).

In some preferred embodiments, the polynucleotide encoding a creatine enzyme is obtained from a polynucleotide sequence encoding a creatine enzyme of alcaligenes origin optimized for synonymous codon usage;

or, the polynucleotide for encoding the creatine enzyme is obtained by optimizing the polynucleotide sequence for encoding the pseudomonas putida source creatine enzyme through synonymous codon preference.

In a second aspect of the invention there is provided an expression vector comprising a polynucleotide provided in the first aspect of the invention.

In some preferred embodiments, the expression vector is an E.coli expression vector, more preferably pET-28a (+).

In a third aspect of the invention there is provided a host cell comprising an expression vector provided in the second aspect of the invention; or alternatively

The host cell has integrated into its genome a polynucleotide as provided in the first aspect of the invention.

In some preferred embodiments, the host cell is E.coli (Escherichia coli).

In some preferred embodiments, the host cell is an E.coli BL21 (DE 3) strain.

In a fourth aspect, the present invention provides a method for preparing creatinase, the method comprising the steps of: culturing the host cell of the third aspect of the invention to express the protein of interest; and

separating the target protein to obtain the creatinase;

wherein, the target protein has an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 3.

In some preferred embodiments, the host cell is obtained by transforming E.coli with a plasmid comprising a polynucleotide according to the first aspect of the invention.

In some preferred embodiments, the host cells are cultured in TB or LB medium. More preferably, the host cells are cultured in TB medium.

In some preferred embodiments, the host cell is cultured in a shaking environment.

In some preferred embodiments, the host cell is cultured at a temperature of 16 to 19 ℃; or culturing the host cell at a temperature of 36 to 38 ℃.

In some more preferred embodiments, higher soluble protein expression of interest is obtained by culturing the host cells in TB medium at a temperature of 16 to 19 ℃.

In some preferred embodiments, the medium used in culturing the host cell comprises a kanamycin resistance gene.

In some preferred embodiments, the host cell is cultured using IPTG to induce expression of the protein of interest.

In some preferred embodiments, the host cell is cultured until an OD600 of 0.6 to 0.8 is reached, and is then induced with IPTG to express the protein of interest.

In some preferred embodiments, the step of isolating the protein of interest comprises:

passing the crushed target protein supernatant through a chromatographic column, eluting, and collecting the eluent.

In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column.

In a fifth aspect, the invention provides a kit comprising: a polynucleotide as provided in the first aspect of the invention; or alternatively

An expression vector as provided in the second aspect of the invention; or alternatively

A host cell according to the third aspect of the invention; or alternatively

Or a creatinase prepared according to the method of the fourth aspect of the present invention.

Compared with the prior art, the invention has at least the following advantages:

(1) The invention develops a creatine enzyme preparation method based on genetic engineering technology, introduces the polynucleotide which codes creatine enzyme and is optimized by synonymous codon preference into a vector, successfully constructs prokaryotic expression plasmid and improves the expression quantity of target protein;

(2) In the preferred embodiment of the invention, the yield of the soluble target protein is further improved by optimizing culture conditions such as culture medium, culture temperature and the like and combining screening of strain sources, and the obtained antigen has high activity.

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

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.

FIG. 1 is a diagram showing SDS-PAGE identification result of creatine enzyme prepared in example according to the present invention;

FIG. 2 is an electrophoresis chart of creatine enzyme obtained by induction expression of a creatine enzyme gene derived from Alcaligenes in accordance with the embodiment of the present invention;

FIG. 3 is an electrophoresis chart of creatine enzyme obtained by induction expression of Pseudomonas putida-derived creatine enzyme gene in accordance with the embodiment of the present invention;

FIG. 4 is a graph showing the standard measurement of the enzymatic activity of creatinase in examples according to the present invention.

Detailed Description

Through extensive and intensive research, the inventor develops a creatine enzyme expression system based on a prokaryotic expression system, and further obtains a polynucleotide sequence for encoding creatine enzyme capable of expressing a large amount of target protein in an escherichia coli expression system through synonymous codon preference optimization, and the expressed target protein has high activity and good stability.

The inventors have further obtained a method for high-efficiency soluble expression of creatine enzyme by optimizing the expression conditions of creatine enzyme, including optimization of the culture medium and culture temperature. In a more preferred embodiment of the invention, host cells comprising a polynucleotide encoding a creatine enzyme of the invention are cultured with TB medium at a temperature of 18 ℃.

Acquisition of a Gene of interest/acquisition of a nucleic acid sequence related to a protein of interest

The full-length nucleotide sequence of the target protein or its element or a fragment thereof of the present invention can be usually obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present invention. 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.

In one embodiment of the present invention, the amino acid sequence of the target protein (SEQ ID NO:1 or SEQ ID NO: 3) is analyzed by NCBI database to obtain the sequence information of the target gene. The amino acid sequence of alcaligenes or pseudomonas putida creatinase is analyzed, for example, by NCBI database, to obtain the target gene sequence encoding the same.

Synonymous codon bias optimization

To overcome the potential problem of reduced yield when expressing heterologous proteins in E.coli, the present invention relates to synonymous codon-biased optimized polynucleotide sequences. The obtained target gene sequence is subjected to synonymous codon preference optimization, and the target gene sequence (SEQ ID NO:2 or SEQ ID NO: 4) subjected to synonymous codon preference optimization can express the same amino acid sequence as the target protein

The invention also relates to polynucleotides having a homology of more than 95% with the sequences shown in SEQ ID NO.2 or SEQ ID NO. 4-7; and a polynucleotide complementary to the sequence shown in SEQ ID No.2 or SEQ ID No. 4-7.

In the present invention, the sequence shown in SEQ ID NO.2 is expressed in a soluble manner as the sequences shown in SEQ ID NO. 4-7, but the sequence shown in the final product SEQ ID NO.2 is not active. While SEQ ID NO.4 has soluble expression compared with the sequences of SEQ ID NO. 5-7, the soluble expression yield is unfavorable, and the soluble expression quantity of SEQ ID NO.4 is obviously higher than that of SEQ ID NO. 5-7.

Vector of target gene

The invention also relates to vectors comprising the polynucleotides of the invention. "vector" in the present invention means a linear or circular DNA molecule comprising a fragment encoding a protein of interest operably linked to other fragments providing for its transcription. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, a vector, and the like. The vector fragment may be derived from the host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, synthetic or conveniently subjected to recombinant DNA procedures, the choice of vector generally being dependent on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the host cell genome and replicates with the chromosome with which it is integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the invention pET-28a is selected as a vector to obtain higher expression efficiency.

Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences of the proteins of the invention and appropriate transcriptional/translational control signals. 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. Illustratively, the insertion of the exogenous DNA fragment is accomplished by cleaving the vector DNA molecule with a DNA endonuclease into a linear molecule that can be linked to the exogenous gene, and then ligating the codon optimized fragment of the gene of interest to the vector, optionally with a single restriction site cohesive end ligation, double restriction site directional cloning, cohesive end ligation of different restriction sites, blunt end ligation, artificial linker ligation, or end ligation with an oligonucleotide.

Transformation of host cells with vectors containing genes of interest

The invention also relates to host cells genetically engineered with the vector or fusion protein coding sequences of the invention. The vector containing the codon-optimized gene of interest may be inserted, transfected or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest. A "host cell" in the present invention is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell may be a eukaryotic host cell or a prokaryotic host cell, the host cell preferably being a bacterium, and preferably being E.coli, more preferably being E.coli BL21 (DE 3) species.

Method for producing target protein

The invention also relates to a method for preparing the target protein, and the polynucleotide sequence can be used for expressing or producing recombinant protein. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Separating and purifying the protein from the culture medium or the cells.

Wherein, the transformation or transduction of the recombinant expression vector containing the polynucleotide of the step (1) into a suitable host cell can be performed by conventional techniques well known to those skilled in the art, and when the host is E.coli, a heat shock method, an electrotransformation method, etc. can be selected.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media, preferably LB, TB media, depending on the host cell used. The culture is carried out 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 order to promote the expression of the target protein and to increase the expression level of the soluble protein, a preferred embodiment of the present invention uses a host cell cultured in TB medium, and the medium used contains a kanamycin resistance gene.

To further promote soluble expression of the protein of interest, in a preferred embodiment of the invention, the host cell is cultured to OD ₆₀₀ After 0.6-0.8 induction with IPTG and further incubation at 17 to 19 ℃ for about 8 to 12 hours.

The protein in the above method may be expressed in the cell, or on the cell membrane, or secreted outside the cell. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In one embodiment of the invention, affinity chromatography is used to molecular target proteins.

In the present disclosure, any exemplary or exemplary language (e.g., ") provided for certain embodiments herein is used merely to better present the disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

If the definition or use of a term in a reference is inconsistent or inconsistent with the definition of that term described herein, the definition of the term described herein applies and the definition of the term in the reference does not apply.

Various terms used herein are shown below. If a term used in a claim is not defined below, the broadest definition persons in the pertinent art have given that term are given as reflected in publications or issued patents that are printed at the time of application.

As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., a nucleic acid or polypeptide) that the nucleic acid or polypeptide is found in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in solvents, buffers, ions or other components that are normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.

As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

The invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

As used herein, the term "codon optimization" refers to a manner of improving the efficiency of gene synthesis by avoiding the use of low-availability or rare codons according to codon usage differences exhibited by organisms (including e.coli, yeast, mammalian blood cells, plant cells, insect cells, etc.) that actually do protein expression or production.

As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is aligned, the number of positions in the aligned polynucleotide or polypeptide that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, according to the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertions or deletions of one or two nucleotides in the polynucleotide). The polypeptides may differ at one position, for example, by containing an amino acid (i.e., substitution or variation) or a deletion of an amino acid (i.e., an amino acid or deletion of an amino acid inserted into one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

As used herein, the terms "sequence complementary" and "reverse sequence complementary" are used interchangeably to refer to a sequence that is opposite in direction to and complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is actaac, then its reverse complement is GTTCAT.

As used herein, the term "expression" includes any step involving the production of a polypeptide in a host cell, including, but not limited to, transcription, translation, post-translational modification, and secretion. After expression, the host cells or expression products can be harvested, i.e.recovered.

The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. 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 experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit the exemplary embodiments of this application.

Example 1 construction of a creatinase plasmid and transfection of host cells

(1) The method comprises the steps of obtaining an amino acid sequence SEQ ID NO. 1 of the alcaligenes creatinase, analyzing the amino acid sequence SEQ ID NO. 1 to obtain a gene sequence, and carrying out synonymous codon preference optimization on the gene sequence to obtain a gene sequence SEQ ID NO.2 subjected to synonymous codon preference optimization. After optimization of the synonymous codon preference of the escherichia coli, the ligation vector is pET-28a to synthesize a recombinant expression plasmid.

The amino acid sequence SEQ ID NO. 3 of pseudomonas putida creatine enzyme is obtained, the pseudomonas putida creatine enzyme is analyzed to obtain a gene sequence, and the gene sequence is subjected to synonymous codon preference optimization to obtain a gene sequence SEQ ID NO.4 subjected to synonymous codon preference optimization. After optimization of the synonymous codon preference of the escherichia coli, the ligation vector is pET-28a to synthesize a recombinant expression plasmid.

(2) Recombinant plasmid introduction into host E.coli

1. Mu.L of the expression plasmid prepared in (1) above was taken and added to 30. Mu.L of E.coli competent BL21 (DE 3) under ice bath conditions, the mixture was left in ice bath for 30min, water bath at 42℃for 45s, immediately left on ice for 2min, 400. Mu.L of antibiotic-free SOC medium was added, and the mixture was subjected to shaking culture at 37℃for 45min at 230 rpm. 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 ℃.

SEQ ID NO:1

MTDDMLHVMKWHNGEKDYSPFSDAEMTRRQNDVRGWMAKNNVDAALFTSYHCINYYSGWLYCYFGRKYGMVIDHNNATTI

SAGIDGGQPWRRSFGDNITYTDWRRDNFYRAVRQLTTGAKRIGIEFDHVNLDFRRQLEEALPGVEFVDISQPSMWMRTIK

SLEEQKLIREGARVCDVGGAACAAAIKAGVPEHEVAIATTNAVIREIAKSFPFVELMDTWTWFQSGINTDGAHNPVTNRI

VQSGDILSLNTFPMIFGYYTALERTLFCDHVDDASLDIWEKNVAVHRRGLELIKPGARCKDIAIELNEMYREWDLLKHRS

FGYGHSFGVLCHYYGREAGVELREDIDTELKPGMVVSMEPMVMLPEGMPGAGGYREHDILIVGEDGAENITGFPFGPEHN

IIRN

SEQ ID NO:2

ATGACTGATGATATGCTGCACGTTATGAAATGGCACAATGGCGAAAAAGATTACTCTCCGTTTAGCGATGCTGAGATGAC

GCGTCGTCAGAACGATGTTCGTGGTTGGATGGCTAAAAACAACGTCGATGCGGCCCTGTTCACCAGCTATCACTGTATTA

ACTACTACTCTGGTTGGCTGTATTGCTATTTCGGCCGTAAGTACGGCATGGTAATCGATCACAACAACGCTACTACCATC

AGCGCGGGCATCGACGGTGGCCAGCCATGGCGTCGTTCCTTCGGCGATAATATCACCTACACTGATTGGCGCCGCGATAA

CTTTTATCGTGCTGTTCGTCAGCTGACGACCGGCGCCAAACGTATTGGCATTGAATTCGACCACGTGAACCTGGACTTCC

GTCGTCAGCTGGAAGAGGCGCTGCCGGGTGTCGAATTCGTTGATATTTCCCAGCCGTCCATGTGGATGCGTACCATTAAG

TCGCTGGAAGAACAGAAACTGATTCGTGAGGGCGCACGCGTGTGTGACGTCGGTGGTGCCGCTTGCGCAGCTGCTATCAA

GGCTGGTGTTCCGGAACATGAGGTCGCGATCGCGACTACTAATGCGGTTATCCGTGAAATCGCGAAAAGCTTCCCATTTG

TGGAACTGATGGACACGTGGACTTGGTTCCAGTCTGGCATCAACACCGACGGCGCGCACAACCCGGTTACCAAGCGTATC

GTTCAGTCTGGTGACATCCTGTCCCTGAACACCTTTCCGATGATCTTCGGCTAATACACCGCGCTGGAACGCACCCTGTT

TTGTGACCACGTGGACGACGCGATCCTGGACATCTGGGAGAAAAACGTTGCCGTGCACCGTCGTGGTCTGGAACTGATCA

AACCGGGTGCTCGTTGTAAAGATATCGCTATCGAACTGAACGAAATGTACCGTGAATGGGACCTGCTGAAACACCGTTCT

TTCGGCTACGGCCACTCCTTCGGTGTTCTGTGTCACTACTACGGCCGTGAAGCTGGCGTGGAACTGCGTGAAGATATCGA

CACCGAACTGAAACCGGGTATGGTAGTTAGCATGGAACCAATGGTCATGCTGCCGGAAGGCATGCCGGGCGCTGGCGGCT

ACCGTGAACATGATATTCTGATTGTCGGTGAAGATGGCGCTGAAAACATCACCGGCTTCCCGTTCGGTCCGGAACATAAC

ATTATCCGCAAC

SEQ ID NO:3

MQMPKTLRIRNGDKVRSTFSAQEYANRQARLRAHLAAENIDAAIFTSYHNINYYSDFLYCSFGRPYALVVTEDDVISISA

NIDGGQPWRRTVGTDNIVYTDWQRDNYFAAIQQALPKARRIGIEHDHLNLQNRDKLAARYPDAELVDVAAACMRMRMIKS

AEEHVMIRHGARIADIGGAAVVEALGDQVPEYEVALHATQAMVRAIADTFEDVELMDTWTWFQSGINTDGAHNPVTTRKV

NKGDILSLNCFPMIAGYYTALERTLFLDHCSDDHLRLWQVNVEVHEAGLKLIKPGARCSDIARELNEIFLKHDVLQYRTF

GYGHSFGTLSHYYGREAGLELREDIDTVLEPGMVVSMEPMIMLPEGLPGAGGYREHDILIVNENGAENITKFPYGPEKNI

IRK

SEQ ID NO:4

ATGCAGATGCCGAAAACCCTGCGTATTCGTAACGGTGATAAAGTTCGTTCCACCTTTTCTGCGCAGGAATACGCGAACCG

TCAGGCGCGTCTGCGCGCTCACCTGGCCGCCGAGAATATTGATGCCGCGATTTTCACTTCCTACCATAACATCAACTACT

ACTCTGACTTCCTGTATTGTAGCTTTGGTCGTCCGTACGCCCTGGTCGTTACTGAAGATGACGTTATTTCTATCTCCGCT

AATATCGATGGTGGTCAGCCGTGGCGTCGCACTGTGGGTACCGATAATATCGTATACACCGATTGGCAGCGCGACAACTA

TTTCGCTGCTATTCAGCAGGCTCTGCCGAAGGCGCGTCGTATCGGTATTGAACACGATCACCTGAACCTGCAAAATCGTG

ACAAACTGGCAGCGCGTTATCCGGACGCAGAACTGGTAGATGTGGCGGCCGCTTGTATGCGTATGCGTATGATCAAATCT

GCAGAAGAACATGTTATGATCCGTCATGGCGCACGCATTGCGGATATCGGTGGTGCCGCCGTTGTGGAAGCACTGGGCGA

CCAGGTCCCGGAATATGAGGTCGCGCTGCACGCAACCCAGGCTATGGTACGTGCGATCGCAGATACCTTCGAGGACGTTG

AACTGATGGACACTTGGACTTGGTTCCAGTCTGGCATCAACACTGATGGTGCACACAACCCGGTTACGACCCGTAAAGTG

AACAAAGGCGATATTCTGTCTCTGAATTGTTTTCCGATGATTGCCGGCTACTATACCGCGCTGGAACGTACCCTGTTCCT

GGACCACTGCTCCGATGACCACCTGCGCGTGTGGCAGGTGAACGTCGAAGTGCACGAAGCGGGTCTGAAGCTGATCAAAC

CGGCCGTCCGCTGCTCCGACATCGCGCGTGAACTGAACGAAATTTTCCTGAAGCACGACGTACTGCAGTACCGCACCTTT

GGCTACGGCCATAGCTTCGGCACTCTGTCCCACTACTACGGTCGTGAAGCCGGCCTGGAACTGCGTGAGGATATCGACAC

CGTTCTGGAACCGGGTATGGTGGTTTCCATGGAACCGATGATTATGCTGCCGGAGGGTCTGCCGGGTGCTGGCTGTTACC

GTGAACACGACATTCTGATCGTGAACGAAAACGGCGCTGAAAACATCACCAAATTCCCGTACGGTCCGGAAAAAAACATC

ATCCGTAAA

Example 2 expression of the Gene of interest

Step 1 the reference preparation of creatinase monoclonal, aseptic operation inoculated into 100 u g/mL Carna resistance TB medium, 37 degrees 220rpm shaking culture to OD600 between 0.6-0.8, IPTG induction, respectively in 37 degrees and 18 degrees shaking culture overnight. Sampling and ultrasonication for SDS-PAGE identification. The identification results are shown in FIG. 1.

As shown in columns 2-4 of FIG. 1, the corresponding codons of Pseudomonas putida-derived creatine enzymes were induced to culture in TB medium at 18℃with soluble background expression.

As shown in columns 5-7 of FIG. 1, the corresponding codon of the creatinase derived from alcaligenes is induced and cultured at 18 ℃ in a TB medium, and the expression of a very small amount of soluble background is carried out, so that the inclusion body is large in proportion.

Example 3 purification of expression products

The recombinant expression strain derived from alcaligenes and the recombinant expression strain derived from pseudomonas putida, which are obtained in the example 2, are respectively inoculated into a TB culture medium for culture, induced expression culture is carried out at 18 ℃, and 4g of thalli are obtained.

20ml of Lysis Buffer was added to the cells and resuspended in ice. Ultrasonic disruption of cells: 10# probe, power 10%, more than 5.5S, stopping 9.9S, and ultrasonic crushing for 30min. Centrifugation was performed at 20000rpm at 4℃for 30min, and the supernatant was collected, filtered through a 0.22 μm membrane, and purified with 1ml of Ni-NTA. The flow rate was 0.5ml/min, and the loading was done using 20ml Lysis Buffer rinse UV and conductance to baseline. The elution procedure included: step 1:0% B,8CV,2ml/min; step 2:0-60% B,20CV,2ml/min; step 3:100% B, 10CV,2ml/min.

Eluent composition:

reagent(s)	BufferA	BufferB	Lysis Buffer
				Tris	50mM	50mM	50mM
NaCl	50mM	50mM	300mM
				Glycerol	-	-	-
Imidazole	-	500mM	-
				pH	7.0	7.0	7.0

The electrophoresis results of the purified sample of the recombinant strain of alcaligenes source creatine enzyme are shown in figure 2. As shown in FIG. 2, from the electrophoresis result and the purification result, the main peak after the purification is the target protein peak, and trace protein passes out during the penetration, so that the purity of the purified target protein is higher.

Taking 21ml of purified sample at peak position, respectively placing into 10KD dialysis bags, respectively placing into 500ml of dialyzate, and dialyzing to obtain volume 21ml. Taking dialyzed samples, diluting for 4 times respectively, measuring concentration by BCA method, R ² Protein concentration was measured to be 1.128mg/ml, yield was 23.688mg,5.922mg/g bacteria =0.999.

The Pseudomonas putida-derived expression product was purified in the same manner, and the result of the sample collection electrophoresis is shown in FIG. 3.

As shown in FIG. 3, the purified sample was collected at the peak position for a total of 12mL. Put into a 10KD dialysis bag and put into 500mL of dialysate for dialysis. Taking dialyzed samples, diluting for 4 times respectively, measuring concentration by BCA method, R ² Sample BCA concentration 3.955mg/ml, yield 47.46mg,11.865mg/g, purified expression product from pseudomonas putida was much higher than purified expression product from alcaligenes.

EXAMPLE 4 identification of target protein Activity by chemiluminescence

(1) Solution preparation

0.2M PBS pH 6.6: firstly, preparing 0.2M mother solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and mixing according to the proportion of 1:1.6.

Creatine solution: 0.1429g creatine powder was weighed, 2.5mL of 0.2M PBS was added, and deionized water was added to a volume of 10mL.

Para-diaminobenzaldehyde solution: 0.2g of p-diaminobenzaldehyde powder was weighed out and 10mL of LDMSO and 1.5mL of hydrochloric acid were added.

5mL of the working fluid was prepared in accordance with Table 1 below

TABLE 1

Pipe number	Diluent volume (mu L)	Creatinase volume and source (μl)	Final concentration of creatine enzyme (U/. Mu.L)
				1	13.6	6.4# stock solution	0.32
2	54	6# stock solution	0.1
				3	4	16#2	0.08
4	7.2	12.8#2	0.064
				5	10	10#2	0.05
6	13.6	6.4#2	0.032
				7	18	2#2	0.01

(2) Experimental procedure

Configuration of positive enzyme (commercially available creatine enzyme positive enzyme, available from Shanghai Seiyaku Biotechnology Co., ltd.; cat# S10182-500U): 500U positive enzyme was dissolved in 0.5mL PBS pH 7.4 buffer (containing 50% glycerol) to prepare 1U/. Mu.L enzyme solution, which was gradually diluted according to the gradient, and the dilution was PBS pH 7.4 buffer.

The enzyme label instrument is preheated for 30min. 100. Mu.L of creatine solution was added to the EP tube, followed by 1. Mu.L of each concentration of enzyme, and reacted at 37℃for 10 minutes. 200. Mu.L of a p-diaminobenzaldehyde solution was then added thereto and incubated at 25℃for 20min. The blank group was not added with enzyme solution.

Program setting of the enzyme label instrument: 100. Mu.L of the reaction solution was pipetted into an ELISA plate and absorbance was measured at 435 nm. The OD difference A2-A1 of the sample and blank was calculated. The results of the creatinase standard curve are shown in FIG. 4.

The absorbance of the purified product of Pseudomonas putida source prepared in the examples of the present invention was measured according to the above method, and the results are shown in Table 2 below:

TABLE 2

According to the results in table 1 above, the stock solution concentration was 3.95mg/mL, the average viability was 0.245U/μl, and the specific viability was (0.245×1000)/3.95=62.03U/mg.

The same method was used to determine the purified product from alcaligenes, and the results showed very low enzyme activity.

Example 5 Pseudomonas putida creatinase Gene codon optimization

In order to improve the expression efficiency and the yield, the gene sequence for encoding the pseudomonas putida source creatine enzyme is subjected to escherichia coli synonymous codon preference optimization in the embodiment, so that a plurality of optimized codons are obtained, and part of optimized codons are shown as SEQ ID NO. 5-7.

SEQ ID NO:5

ATGCAGATGCCTAAAACACTGCGTATTCGCAATGGGGACAAAGTTCGTTCCACATTTTCTGCCCAGGAATATGCAAACCG

GCAGGCCCGTTTACGGGCACATTTAGCCGCTGAAAATATTGATGCAGCTATCTTTACCAGCTACCATAATATTAACTACT

ACAGCGATTTTCTTTACTGTTCGTTTGGACGTCCATATGCTCTTGTTGTTACGGAAGACGATGTCATTTCTATCTCAGCA

AACATCGATGGCGGTCAACCTTGGAGACGTACGGTAGGTACAGATAATATTGTCTATACAGACTGGCAGCGGGATAATTA

TTTTGCGGCTATCCAACAAGCACTCCCTAAAGCACGACGGATTGGGATTGAGCACGACCATCTGAATTTGCAAAATCGTG

ATAAACTTGCAGCTCGCTATCCAGATGCTGAACTTGTCGATGTAGCCGCAGCCTGCATGCGTATGCGCATGATCAAATCA

GCGGAAGAACATGTTATGATTAGACATGGAGCGCGTATTGCTGATATTGGGGGCGCTGCAGTCGTTGAAGCGCTGGGAGA

TCAAGTTCCGGAATATGAGGTTGCCCTGCATGCGACACAAGCAATGGTTAGAGCTATCGCCGACACGTTTGAAGACGTTG

AATTGATGGATACTTGGACATGGTTTCAGTCGGGAATTAATACGGATGGGGCCCATAATCCAGTTACAACGAGAAAAGTT

AACAAAGGCGATATATTGTCGCTGAATTGCTTCCCGATGATTGCAGGCTACTACACTGCACTTGAGCGTACCCTGTTTTT

GGATCACTGCTCAGATGATCATCTCCGTCTTTGGCAAGTCAATGTTGAAGTCCACGAAGCTGGCCTTAAGTTGATCAAAC

CTGGGGCGCGGTGCTCAGATATTGCCAGAGAATTAAACGAAATCTTTCTGAAACATGACGTCCTCCAGTATAGAACATTT

GGCTATGGCCATAGTTTCGGCACACTGTCCCACTATTATGGAAGAGAAGCCGGTCTTGAGCTTCGGGAAGACATCGATAC

CGTACTGGAGCCAGGCATGGTAGTTAGCATGGAGCCAATGATCATGTTACCGGAGGGATTGCCGGGGGCAGGCGGCTATA

GAGAGCATGATATTCTTATCGTCAATGAAAACGGGGCAGAAAATATTACAAAGTTCCCGTACGGGCCTGAGAAGAACATC

ATCCGTAAA

SEQ ID NO:6

ATGCAAATGCCTAAGACATTAAGAATTAGAAATGGTGATAAGGTCCGAAGTACTTTTTCTGCCCAAGAATACGCCAACAG

ACAAGCCAGACTGAGAGCCCACCTGGCTGCTGAGAACATAGATGCTGCTATTTTTACATCTTATCACAACATCAATTATT

ACTCTGATTTCTTGTACTGTTCTTTCGGAAGACCTTACGCTCTGGTAGTAACGGAGGACGATGTTATCTCTATCAGTGCT

AATATCGACGGTGGACAACCATGGAGAAGAACTGTTGGAACCGATAACATTGTGTATACCGATTGGCAAAGGGATAATTA

CTTCGCTGCAATTCAACAAGCTCTACCCAAAGCTAGAAGAATTGGAATTGAACATGATCACCTAAACTTGCAGAACAGAG

ATAAATTGGCAGCTAGATATCCAGATGCTGAATTAGTTGACGTGGCTGCAGCATGTATGAGAATGAGAATGATTAAGTCC

GCAGAAGAACACGTGATGATTAGACATGGAGCACGAATTGCAGACATTGGCGGTGCCGCTGTTGTTGAAGCCTTAGGCGA

CCAAGTCCCAGAATACGAGGTTGCCTTACATGCTACTCAAGCAATGGTGCGTGCCATTGCCGATACTTTTGAAGACGTTG

AACTTATGGACACTTGGACTTGGTTTCAATCAGGTATCAATACAGACGGAGCACATAACCCCGTTACAACCAGAAAGGTT

AACAAAGGTGATATTTTGTCTTTGAATTGTTTCCCAATGATTGCTGGATACTACACCGCTTTGGAAAGGACGCTTTTCTT

GGATCATTGTTCAGACGACCACCTTAGGTTATGGCAGGTGAACGTCGAGGTACATGAGGCTGGTCTTAAGCTAATTAAAC

CAGGTGCTCGTTGCTCCGACATCGCACGTGAACTTAACGAGATTTTCCTTAAACATGATGTATTGCAATACCGTACTTTC

GGTTATGGTCATTCATTTGGTACCCTTTCTCACTATTACGGCCGAGAGGCCGGATTGGAGTTGCGAGAGGACATTGACAC

TGTTCTGGAACCTGGAATGGTTGTTTCTATGGAACCTATGATCATGCTACCAGAAGGATTGCCAGGTGCCGGAGGATATA

GGGAGCATGATATTTTAATCGTTAACGAAAATGGAGCCGAAAACATCACTAAATTTCCTTATGGACCTGAGAAGAATATT

ATAAGAAAA

SEQ ID NO:7

ATGCAGATGCCAAAGACCCTGCGAATCAGGAACGGTGACAAAGTGCGGTCAACATTTTCTGCTCAGGAGTATGCAAACCG

CCAGGCAAGACTCAGAGCTCATCTGGCAGCTGAGAATATCGACGCAGCCATTTTCACAAGTTATCATAATATCAATTACT

ATTCCGACTTCTTGTACTGCTCATTCGGCAGACCTTACGCCCTGGTCGTCACGGAGGACGATGTCATCAGCATTTCCGCA

AACATTGATGGAGGACAACCATGGAGACGGACAGTGGGCACTGACAACATCGTCTACACAGATTGGCAACGAGATAATTA

CTTTGCAGCAATCCAGCAGGCCCTGCCAAAAGCTCGCAGGATAGGGATAGAGCACGATCACTTGAATCTTCAAAACAGAG

ATAAGCTGGCAGCTCGGTATCCCGACGCTGAGTTGGTGGATGTAGCGGCCGCATGCATGCGCATGCGGATGATAAAAAGC

GCTGAGGAGCACGTAATGATTCGGCATGGGGCAAGGATTGCCGACATTGGAGGTGCTGCCGTGGTAGAGGCTCTGGGAGA

TCAGGTTCCTGAATATGAAGTTGCTCTCCACGCAACCCAAGCTATGGTCAGGGCTATCGCCGACACCTTCGAGGACGTTG

AGCTGATGGACACCTGGACCTGGTTTCAAAGCGGCATCAATACAGATGGCGCTCATAACCCCGTGACTACCCGGAAGGTT

AACAAAGGAGATATCCTGAGCCTGAATTGCTTTCCAATGATCGCTGGCTACTATACCGCGCTGGAGCGGACCCTGTTTCT

GGATCACTGCTCCGACGACCATTTGCGCCTGTGGCAGGTGAACGTGGAAGTGCATGAAGCAGGCCTTAAGCTGATCAAAC

CTGGAGCTAGGTGTTCCGATATTGCCAGAGAGCTCAACGAGATTTTTCTGAAGCACGATGTGCTGCAGTACCGGACTTTC

GGCTACGGACACTCATTTGGCACGCTGAGCCATTATTACGGACGCGAAGCAGGCTTGGAACTCAGAGAGGATATCGACAC

CGTGTTGGAGCCCGGGATGGTGGTGAGCATGGAGCCAATGATTATGCTTCCTGAAGGCTTGCCTGGCGCTGGCGGTTATA

GAGAGCACGATATTCTGATAGTCAATGAGAACGGCGCCGAAAATATCACCAAGTTTCCTTATGGACCCGAGAAAAATATA

ATTAGGAAA

Plasmids were constructed and E.coli transformed in the same manner as in example 1, and the monoclonal was cultured and carried out under the same culture conditions as in example 2, and purification was carried out under the conditions as in example 3, whereby the yields of the target proteins were recorded in Table 3 below.

TABLE 3 Table 3

Codons	Sequence(s)	Soluble expression level
			Codon IV	SEQ ID NO:4	90％
Codon V	SEQ ID NO:5	70％
			Codon VI	SEQ ID NO:6	60％
Codon VII	SEQ ID NO:7	40％

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

1. An isolated polynucleotide encoding a creatinase, wherein the polynucleotide is codon optimized and the polynucleotide is selected from any one of the following:

(i) A polynucleotide having a sequence as shown in SEQ ID NO.2 or SEQ ID NO. 4;

2. The polynucleotide according to claim 1, wherein the polynucleotide encoding a creatine enzyme is obtained from a polynucleotide sequence encoding a pseudomonas putida source creatine enzyme by synonymous codon bias optimization;

3. An expression vector comprising the polynucleotide of claim 1 or 2.

4. The expression vector according to claim 3, characterized in that the expression vector is an E.coli expression vector, preferably pET-28a.

5. A host cell comprising the expression vector of any one of claims 3 or 4; or alternatively

The polynucleotide according to claim 1 or 2 integrated into the genome of the host cell.

6. A method of preparing creatinase, the method comprising the steps of:

transforming a host cell with a vector comprising the polynucleotide of claim 1 or 2;

culturing the host cell to express the creatinase.

7. The method of claim 5, wherein the host cells are cultured in LB or TB medium;

and/or, when the host cell is cultured, the medium used contains a kanamycin resistance gene.

And/or, when the host cell is cultured, the target protein is expressed by IPTG induction.

And/or culturing the host cell at a temperature of 16 to 19 ℃ while culturing the host cell.

And/or, when culturing the host cell, culturing to an OD600 of 0.6 to 0.8, and then inducing with IPTG to express the target protein.

8. The method of claim 5, wherein said step of culturing said host cell is followed by the step of:

separating the target protein to obtain the creatinase.

9. The method of claim 8, wherein the step of isolating the protein of interest comprises:

10. A kit, comprising: the polynucleotide of claim 1 or 2; or alternatively

The expression vector of any one of claims 3 or 4; or alternatively

The host cell of claim 5; or alternatively

Creatinase produced by the method of any one of claims 6 to 9.