阅读说明：本技术 一种来源于单增李斯特菌的重组氨基肽酶t及应用 (Recombinant aminopeptidase T derived from Listeria monocytogenes and application thereof ) 是由 程昌勇 宋厚辉 孙静 于 2019-10-08 设计创作，主要内容包括：本发明涉及生物工程技术领域,旨在提供一种来源于单增李斯特菌的重组氨基肽酶T及应用。该氨基肽酶T的氨基酸序列如SEQ ID NO.1所示。用于编码前述氨基肽酶T的基因的核苷酸序列如SEQ ID NO.2所示。本发明获得的重组氨基肽酶T(Lmo1603编码的蛋白)具有氨基肽酶活性,并且对部分金属离子具有较强依赖性。基于此特性,本发明获得的重组氨基肽酶T能够用于蛋白质的深度水解。(The invention relates to the technical field of bioengineering, and aims to provide a recombinant aminopeptidase T derived from Listeria monocytogenes and application thereof. The amino acid sequence of the aminopeptidase T is shown as SEQ ID NO. 1. The nucleotide sequence of the gene for encoding the aforementioned aminopeptidase T is shown in SEQ ID NO. 2. The recombinant aminopeptidase T (protein encoded by Lmo 1603) obtained by the invention has aminopeptidase activity and has strong dependence on partial metal ions. Based on this property, the recombinant aminopeptidase T obtained by the present invention can be used for deep hydrolysis of proteins.)

1. A recombinant aminopeptidase T derived from Listeria monocytogenes, wherein the amino acid sequence of the aminopeptidase T is shown in SEQ ID No. 1.

2. The gene encoding the recombinant aminopeptidase T of claim 1, wherein the nucleotide sequence of the gene is represented by SEQ ID NO. 2.

3. The method for producing the gene according to claim 2, comprising: taking the genome DNA of Listeria monocytogenes EGD-e NC-003210 as a template, and carrying out gene amplification by using polymerase chain reaction to obtain a target gene; the primers used in the amplification procedure were as follows:

an upstream primer: GCGCATATGAGAGATGCAAGAATCGAAAAATTAGCA

A downstream primer: CCGCTCGAGTACTAAGTTTTCTGGATTAAGTGCTTCTAATTCT

Wherein, the underlined part of the upstream primer is Nde I restriction site, and the underlined part of the downstream primer is Xho I restriction site.

4. The method of claim 3,

the PCR system used for amplification was:

KOD plus buffer 10. mu.l; 2 μ l each of the forward primer and the reverse primer, 10 mM; DNA template 2. mu.l and ddH₂O66μl；MgSO₄6μl；dNTP 10μl；KOD-plus-Neo 2μl；

The PCR amplification step is as follows: (1) pre-denaturation at 94 ℃ for 2 min; (2) denaturation at 98 ℃ for 10 s; (3) annealing at 56 ℃ for 30 s; (4) extending for 1min at 68 ℃; repeating the steps (2) to (4) for 30 times; (5) continuing to extend for 7min at 68 ℃, and cooling to 4 ℃; the PCR product was identified by agarose gel electrophoresis.

5. The process for producing a recombinant expression vector of the recombinant aminopeptidase T gene of claim 2, which comprises:

performing double enzyme digestion on the gene of aminopeptidase T for 2h by using restriction enzymes Nde I and Xho I at 37 ℃, purifying by agarose gel electrophoresis, and recovering a target fragment by using an agarose gel DNA recovery kit; connecting the target fragment with pET30 which is also cut by Nde I Xho I under the action of T4DNA ligase for 4-5h at 16 ℃; then transforming to Escherichia coli E.coli DH5 alpha competent cells, screening positive clone by bacteria liquid PCR, extracting plasmid, namely the recombinant expression vector of aminopeptidase T gene, and naming the recombinant expression vector as pSL 327.

6. The process for producing a transformant expressing a recombinant aminopeptidase T gene as claimed in claim 2, which comprises:

(1) preparation of recombinant expression vectors

Performing double enzyme digestion on the gene of aminopeptidase T for 2h by using restriction enzymes Nde I and Xho I at 37 ℃, purifying by agarose gel electrophoresis, and recovering a target fragment by using an agarose gel DNA recovery kit; connecting the target fragment with pET30 plasmid which is also cut by Nde I Xho I under the action of T4DNA ligase for 4-5h at 16 ℃; then transforming to Escherichia coli E.coli DH5 alpha competent cells, screening positive clone by bacteria liquid PCR, extracting plasmid, namely the recombinant expression vector of aminopeptidase T gene, and naming as pSL 327;

(2) preparation of recombinant expression transformant

The plasmid of the recombinant expression vector is transformed into an escherichia coli e.coli.rosetta competent cell, a positive recombinant is screened on a resistance plate containing kanamycin, a monoclonal is selected, and a recombinant expression transformant is obtained after culture and named as e.coli.rosetta-pSL 327.

7. The process for producing recombinant aminopeptidase T according to claim 1, which comprises:

(1) preparation of recombinant expression vectors

Performing double enzyme digestion on the gene of aminopeptidase T for 2h by using restriction enzymes Nde I and Xho I at 37 ℃, purifying by agarose gel electrophoresis, and recovering a target fragment by using an agarose gel DNA recovery kit; connecting the target fragment with pET30a plasmid which is also cut by Nde I Xho I under the action of T4DNA ligase for 4-5h at 16 ℃; then transforming to Escherichia coli E.coli DH5 alpha competent cells, screening positive clone by bacteria liquid PCR, extracting plasmid, namely the recombinant expression vector of aminopeptidase T gene, and naming as pSL 327;

(2) preparation of recombinant expression transformant

Transforming the plasmid of the recombinant expression vector into an Escherichia coli E.coli.Rosetta competent cell, screening a positive recombinant on a resistance plate containing kanamycin, selecting monoclonal recombinant Escherichia coli, and culturing to obtain a recombinant expression transformant, namely E.coli.Rosetta-pSL 327;

(3) expression of recombinant aminopeptidase

Selecting the cultured monoclonal recombinant escherichia coli to fall into 5mL of Kana resistant LB liquid containing 100 mu g/mL, and shaking for overnight culture at 37 ℃; transferring fresh bacterial liquid to a sterile 500mL LB liquid culture medium at a ratio of 1:100, performing shake culture at 37 ℃ until OD600nm is 0.6-0.8, and adding IPTG with a final concentration of 0.6 mM; culturing at 37 deg.C for 4-5h to induce Lmo1603, centrifuging at 4 deg.C 3700rpm/min for 15min, and collecting thallus; 10mM PBS (pH 7.4) was added, the cells were washed once with a heavy suspension, and 20mL PBS (pH 7.4) and 100mM was added; carrying out ultrasonic crushing for 25min for 8s at an interval of 10 s; collecting protein solution, centrifuging at 4 deg.C and 12000rpm/min for 25min, collecting supernatant and precipitate, respectively, 50 μ L each, performing SDS-PAGE analysis, and staining with Coomassie brilliant blue;

(4) protein purification of aminopeptidase T

Adding 2mL of nickel column into the collection tube, washing the nickel column with 20mL of ddH20 after ethanol completely flows out, and then balancing the column with 10mL of 100mM PBS (pH 7.4); adding 40mL of PBS (phosphate buffer solution) with the concentration of 100mM and the pH value of 7.4 to resuspend the Rosetta thalli, and carrying out ultrasonic disruption for 25min at the interval of 10s and 8 s; collecting protein solution, centrifuging at 4 deg.C and 12000rpm/min for 25min, and transferring supernatant to new centrifuge tube; turning and combining the supernatant and the balanced nickel column at 4 ℃ for 3-4h, transferring the combined solution to a collecting column after the combination is finished, adding 30mL of 50mM imidazole to wash out the foreign proteins after the combined solution flows out, and keeping the flow rate at 0.5-1 mL/min; adding 5mL of 400mM imidazole to elute the target protein, and keeping the flow rate at 0.5-1 mL/min; measuring protein concentration with enzyme-labeling instrument, adding purified protein into 100% glycerol, and storing at-20 deg.C.

8. Use of the recombinant aminopeptidase T of claim 1 as a proteolytic enzyme.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to recombinant aminopeptidase T derived from Listeria monocytogenes and application thereof.

Background

Aminopeptidases (APs) are a metalloproteolytic enzyme widely present in animals, plants and microorganisms. Aminopeptidases recognize the N-terminal amino acid residue of a polypeptide and hydrolyze to produce free amino acids. Such as the most common leucine aminopeptidase, can hydrolyze leucine residues at the N-terminus of the polypeptide to produce free leucine and the polypeptide. Aminopeptidases have a variety of functions in eukaryotic cells. Such as processing, maturation and transformation of protein, regulation and control of polypeptide hydrolysis, gene expression and the like. In addition to the above functions, aminopeptidases are also present in the transport mechanism. For example, the ABC polypeptide transporter system (PepTs) in Helicobacter pylori (Helicobacter pylori), the transporters Dpp and Opp may mediate the transporter polypeptide; meanwhile, aminopeptidase PepA participates in the transport of effector proteins, and can specifically hydrolyze the effector proteins, which is important for the survival and infection of bacteria in host cells; helicobacter pylori is able to survive in gastric juices and is primarily responsible for the presence of acid resistance, the main energy sources in the host cell being alanine (L-alanine), serine (L-serine) and other related 6D-amino acids. Like intracellular parasitic bacteria, listeria monocytogenes also require effector agents to facilitate adhesion erosion and intracellular proliferation, aminopeptidases to assist in effector processing and maturation, aminopeptidases to provide a source of nutrients for bacterial intracellular survival, etc., but this mechanism has not been proven.

The genes annotated for aminopeptidase in the listeria monocytogenes EGD-e genome are: lmo1780(peptM20.003), lmo2338(pepC 01.086), lmo1709(map M24A), lmo1578(M24.008), lmo1354(M24B), lmo1711(M29.004) and lmo1603 (unknown) (http:// www.uniprot.org /). Although annotated as aminopeptidase activity, the specific function has not been confirmed by studies.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a recombinant aminopeptidase T derived from Listeria monocytogenes and application thereof.

Compared with the prior art, the solution of the invention is as follows:

provides an aminopeptidase T derived from Listeria monocytogenes, and the amino acid sequence of the aminopeptidase T is shown in SEQ ID NO. 1.

The invention also provides a gene for coding the aminopeptidase T, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.

The aminopeptidase T gene is prepared by the following method:

an upstream primer and a downstream primer (SEQ ID NO.3 and SEQ ID NO.4) are designed and synthesized according to the lmo1603 gene sequence of Listeria monocytogenes (Listeria monocytogenes) EGD-e NC-003210 recorded in Genbank, and then the genome DNA of Listeria monocytogenes (Listeria monocytogenes) EGD-e NC-003210 is used as a template to carry out gene amplification by using Polymerase Chain Reaction (PCR) so as to obtain a target gene.

The primers used in the amplification procedure were as follows:

an upstream primer: GCGCATATGAGAGATGCAAGAATCGAAAAATTAGCA

A downstream primer: CCGCTCGAGTACTAAGTTTTCTGGATTAAGTGCTTCTAATTCT

Among them, the underlined part of the upstream primer (SEQ ID NO.3) is an Nde I cleavage site, and the underlined part of the downstream primer (SEQ ID NO.4) is an Xho I cleavage site.

The base sequence of aminopeptidase T gene is shown in a sequence SEQ ID NO.2, is named as Lmo1603 and has a full length of 1113 bp. Wherein the coding sequence (CDS) is from 1 st base to 1113 rd base, the initiation codon is ATG, and the termination codon is TAA. The amino acid sequence of the encoded protein is shown as SEQ.1 in the sequence table.

The present invention further relates to a recombinant expression vector comprising the nucleotide sequence of the aminopeptidase T gene of the present invention. It can be constructed by ligating the nucleotide sequence of the aminopeptidase T gene of the present invention to various vectors by a method conventional in the art. The vector may be any vector conventionally used in the art, such as a commercially available plasmid, cosmid, phage or viral vector, and the like, and pET30a is preferred. Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the aminopeptidase T gene product obtained by PCR amplification and an expression vector pET30a were subjected to double digestion with restriction enzymes Nde I and Xho I to form complementary cohesive ends, and the cohesive ends were ligated by T4DNA ligase to form a recombinant expression plasmid pSL327 containing the aminopeptidase T gene of the present invention.

The present invention further relates to a recombinant expression transformant comprising the aminopeptidase T gene of the present invention or a recombinant expression vector thereof. It can be prepared by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism may be any of various host microorganisms conventionally used in the art, as long as it is sufficient that the recombinant expression vector can stably replicate by itself and that the aminopeptidase T gene of the present invention carried thereby can be efficiently expressed. Coli, dh5 α and escherichia coli, rosetta (DE3) are preferred in the present invention. The recombinant expression plasmid pSL327 is transformed into Escherichia coli E.coli.Rosetta (DE3), so as to obtain the preferred genetic engineering strain of the invention, i.e., Escherichia coli E.coli.Rosetta (DE3) -pSL 327.

The present invention still further relates to a process for the preparation of recombinant aminopeptidase T comprising: culturing the recombinant expression transformant of the present invention to obtain recombinant aminopeptidase T.

Wherein, the medium used in said culturing the recombinant expression transformant may be any medium in the art that allows the transformant to grow and produce the aminopeptidase T of the present invention, and for the strain, LB medium is preferred: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 7.0 of pH. The culture method and culture conditions are not particularly limited, and may be appropriately selected according to the ordinary knowledge in the art depending on the type of host, the culture method, and the like, as long as the transformant can grow and produce aminopeptidase T. Other specific procedures for culturing the transformant can be performed according to the routine procedures in the art. For the strain, the following method is preferred: recombinant Escherichia coli expressing the aminopeptidase T gene of the present invention (preferably, recombinant expression transformant of Escherichia coli E. coli. Rosetta (DE3) of the present invention) was inoculated into LB medium containing kanamycin and cultured, and when the optical density OD600 of the culture medium reached 0.5 to 0.8, the recombinant aminopeptidase T of the present invention was expressed with high efficiency under the induction of IPTG at a final concentration of 0.6 mM.

The present invention further provides the use of the aforementioned recombinant aminopeptidase T as a proteolytic enzyme.

Compared with the prior art, the invention has the beneficial effects that:

the recombinant aminopeptidase T (protein encoded by Lmo 1603) obtained by the invention has aminopeptidase activity and has strong dependence on partial metal ions. Based on this property, the recombinant aminopeptidase T obtained by the present invention can be used for deep hydrolysis of proteins.

Drawings

FIG. 1 is an electrophoretogram of gene lmo1603 fragment;

FIG. 2 is a schematic diagram of the structure of the cloning plasmid pSL 327;

FIG. 3 shows PCR-screened positive clones in the process of preparing recombinant expression vectors;

FIG. 4 is an electrophoretogram of aminopeptidase T after purification.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The material sources in the following examples are:

pET30a expression plasmids, e.coli.dh5 α and e.coli.rosetta (DE3) competent cells were purchased from shanghai yubo biotechnology limited. KOD plus Neo (PCR kit) was purchased from Shanghai Toyobo; the PCR product purification/recovery kit is purchased from Shanghai Lufeng Biotech limited; restriction endonucleases were purchased from NEB corporation; t4 nucleic acid ligase was purchased from Takara; the plasmid small-extraction kit is purchased from Shanghai Biotechnology Limited; protein chromatography coupled columns (nickel columns) were purchased from Beijing Wai Bohui chromatography technologies, Inc.