CN-122012439-A - Catechol-O-methyltransferase mutant and preparation method and application thereof

CN122012439ACN 122012439 ACN122012439 ACN 122012439ACN-122012439-A

Abstract

The invention provides a catechol-O-methyltransferase mutant, a preparation method and application thereof, and relates to the technical field of biology. The catechol-O-methyltransferase mutant contains 79P mutation relative to wild catechol-O-methyltransferase, and can be expressed in vitro by recombinant cells, thereby solving the problems of poor stability and high cost of the catechol-O-methyltransferase preparation process in the prior art.

Inventors

LI GUILIN
WANG QILEI
MENG QIANQIAN
LI XIN
XU YANWEI
ZHAO QIAOHUI
FU GUANGYU
YANG ZENGLI
WU XUEWEI

Assignees

郑州伊美诺生物技术有限公司

Dates

Publication Date: 20260512
Application Date: 20260226

Claims (10)

1. A catechol-O-methyltransferase mutant comprising a mutation of 79P relative to a wild-type catechol-O-methyltransferase, wherein the position of the amino acid residue is determined using the amino acid sequence shown in SEQ ID NO.1 as a reference sequence.
2. The catechol-O-methyltransferase mutant of claim 1, wherein said wild-type catechol-O-methyltransferase comprises a catechol-O-methyltransferase from a mammal; Alternatively, the wild-type catechol-O-methyltransferase comprises a catechol-O-methyltransferase derived from swine; Optionally, the amino acid sequence of the wild catechol-O-methyltransferase comprises a sequence as shown in SEQ ID No. 1; alternatively, the mutation relative to wild-type catechol-O-methyltransferase is 79P; optionally, the catechol-O-methyltransferase mutant comprises an amino acid sequence shown as SEQ ID NO.2, or comprises an amino acid sequence which has at least 90% identity with the amino acid sequence shown as SEQ ID NO.2, and amino acid residues at positions 19, 56, 116, 132, 141, 171, 183, 186 and 190 of the amino acid sequence are N, Q, A, T, W, C, G, C and T respectively; Optionally, the catechol-O-methyltransferase mutant has an amino acid sequence shown in SEQ ID NO. 2.
3. Catechol-O-methyltransferase mutant according to claim 1 or 2, characterized in that a marker protein is also attached; Optionally, the marker protein comprises a tag protein; Optionally, the tag protein comprises one or more of His tag, GST tag, flag tag, SUMO tag, MBP tag and Strep tag, preferably His tag.
4. A biomaterial, characterized by being selected from any one of (i) to (iii): (i) A polynucleotide encoding the catechol-O-methyltransferase mutant of any one of claims 1-3; (ii) A vector carrying the polynucleotide of (i); (iii) A recombinant cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the catechol-O-methyltransferase mutant of any one of claims 1-3.
5. A method for producing a catechol-O-methyltransferase mutant according to any one of claims 1 to 3, wherein the recombinant cell according to claim 4 is cultured and the catechol-O-methyltransferase mutant is isolated.
6. The method of claim 5, comprising expressing the catechol-O-methyltransferase mutant using a baculovirus-insect cell expression system; Alternatively, the preparation method comprises constructing a baculovirus vector and performing amplification culture on the baculovirus vector until at least a third generation baculovirus vector is obtained, and then using the at least third generation baculovirus vector to infect insect cytopathy amplification culture, so as to obtain the catechol-O-methyltransferase mutant.
7. The method according to claim 5 or 6, wherein the method further comprises isolating and purifying the catechol-O-methyltransferase mutant expressed by insect cells, and/or the method further comprises freeze-drying the catechol-O-methyltransferase mutant; Optionally, the freeze-dried protectant comprises trehalose and the excipient comprises mannitol; Optionally, the working concentration of the trehalose is 0.5-2% w/v, and/or the working concentration of the mannitol is 1-10% w/v; optionally, the trehalose is at a working concentration of 1% w/v and/or the mannitol is at a working concentration of 5% w/v; Optionally, said isolating and purifying said catechol-O-methyltransferase mutant expressed by an insect cell comprises the steps of: (A) Resuspending the cell pellet with a first buffer, then sonicating, collecting the supernatant and filtering, collecting the filtrate; (B) Purifying the filtrate collected in step (a) by chromatography, equilibrating the chromatography column with a second buffer, eluting the sample with a third buffer; (C) Performing ultrafiltration liquid exchange on the eluent in the step (B) by using an ultrafiltration hollow fiber membrane, wherein the liquid exchange is a fourth buffer solution; the first buffer solution, the second buffer solution, the third buffer solution and the fourth buffer solution respectively and independently contain trehalose and/or mannitol.
8. Use of the catechol-O-methyltransferase mutant of any one of claims 1 to 3, or the biomaterial of claim 4, or the preparation method of any one of claims 5 to 7 in any one of (I) to (III): (I) Detecting catecholamines; (II) preparing a test catecholamine product; (III) preparing a product for detecting a condition associated with an abnormality of catecholamines or with an abnormality of catecholamine metabolites; optionally, the catecholamine includes one or more of epinephrine, norepinephrine, isoproterenol, and dopamine; Alternatively, the disorder associated with catecholamine abnormalities or catecholamine metabolite abnormalities comprises secondary hypertension or pheochromocytoma.
9. A kit for catecholamine detection, comprising the catechol-O-methyltransferase mutant according to any one of claims 1 to 3; optionally, the catechol-O-methyltransferase mutant in the kit is a lyophilized formulation; alternatively, the catechol-O-methyltransferase mutant in the kit is a lyophilized formulation obtained according to the preparation method of claim 7.
10. A method for detecting catecholamine, comprising methylating catecholamine in a sample using the catechol-O-methyltransferase mutant according to any one of claims 1 to 3.

Description

Catechol-O-methyltransferase mutant and preparation method and application thereof Technical Field The invention relates to the technical field of biology, in particular to a catechol-O-methyltransferase mutant, a preparation method and application thereof. Background The following statements merely provide background information related to the present disclosure and may not necessarily constitute prior art. Catecholamines are the generic names of epinephrine, norepinephrine, dopamine, etc., produced and released mainly by the sympathetic nerve and adrenal medulla. Adrenal medulla produces mainly epinephrine and small amounts of norepinephrine, and the sympathetic nerves secrete mainly norepinephrine, both of which are eventually excreted from urine as esters by the action of monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) via two different metabolic pathways, 3-methoxy-4-hydroxy mandelic acid (VMA, also known as vanillic acid) in combination with glucuronic acid or sulfuric acid. In pathological conditions, especially tumours of chrophilic tissue, secretion produces large amounts of epinephrine and norepinephrine, causing an increase in blood pressure. COMT is one of the major mammalian enzymes of catecholamines involved in the metabolic degradation of catecholamine transmitters (e.g., epinephrine, norepinephrine, isopropanolamine, dopamine, etc.) in the body, and acts by transferring the methyl group on the methyl donor S-adenosylmethionine (SAM) to the hydroxyl groups at the 3-and 4-positions of catecholamine, thereby inactivating the catecholamine neurotransmitters. In addition to playing a role in the metabolism of endogenous substances, COMT is a key target in catechol drug development for hypertension, asthma, and parkinson's disease. COMT exists widely in a variety of mammalian tissues in the form of membrane-bound and secreted 2 forms, which differ in molecular sequence and function, and in tissue distribution. COMT plays an important role in the field of medical inspection, and particularly in the research of developing catecholamine three (dopamine, epinephrine, norepinephrine) detection kits, the physiological component indexes related to diseases such as secondary hypertension, pheochromocytoma and the like are monitored by converting catecholamine substances into methylated substances which are more beneficial to analysis and detection. At present, the diagnosis kit mainly adopts COMT of a natural extraction process, the natural extraction process mainly adopts fresh pork liver to carry out COMT separation and purification and finished product freeze-drying, and the process steps comprise pork liver acquisition, pork liver tissue breaking, cell lysis and leaching, clarification after acidification, ammonium sulfate precipitation, ultrafiltration concentration liquid exchange, chromatographic separation and preparation freeze-drying. The main defects of the process are 1) pig liver batch and source control, namely that the problem of larger COMT content difference in pig livers of different batches exists in the process due to the fact that the sources of pig livers cannot be effectively controlled, the stability of the subsequent preparation process is reduced, 2) separation and purification process control, namely that an acid precipitation process and an ammonium sulfate precipitation process are needed, the addition of two-step primary purification process leads to longer process route and increases the instability risk in the enzyme preparation process, 3) finished product preparation process control, namely that components are directly applied as a kit, a prescription is selected as a basic formula, and the appearance control of a finished product in freeze drying is needed to be improved, and 4) the whole production process cost control, namely that the process route is long, the raw material, consumable and time cost are improved, the process yield is lower, and the estimated production cost is more than 200 yuan/mg. Therefore, there is a need to improve the stability of COMT manufacturing processes and reduce production costs. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a catechol-O-methyltransferase mutant and a preparation method thereof, so as to solve the problems of poor stability and high cost of catechol-O-methyltransferase in the preparation process in the prior art. In order to solve the technical problems, the invention adopts the following technical scheme: In a first aspect, a catechol-O-methyltransferase mutant is provided, comprising a mutation of 79P relative to a wild-type catechol-O-methyltransferase, wherein the position of the amino acid residue is determined using the amino acid sequence shown in SEQ ID NO.1 as a reference sequence. In a second aspect, a biomaterial is provided, the biomaterial being selected from any one of (i) - (iii): (i) A polynucleotide encoding the catechol-O-methy