CN118308328A

CN118308328A - Mannosidase mutant and recombinant gene thereof

Info

Publication number: CN118308328A
Application number: CN202410566477.XA
Authority: CN
Inventors: 刘德川; 樊兵; 马睿
Original assignee: Hangzhou Liwen Institute Biotechnology Co ltd
Current assignee: Hangzhou Liwen Institute Biotechnology Co ltd
Priority date: 2024-05-09
Filing date: 2024-05-09
Publication date: 2024-07-09

Abstract

The invention discloses a mannosidase mutant and a recombinant gene thereof. The mannosidase mutant has better enzyme digestion activity on alpha 1-2, alpha 1-3 and alpha 1-6. The mannosidase mutant has the same enzyme activity on alpha 1-2 glycosidic bonds as the wild type, and has obviously improved enzyme activity on alpha 1-3 and alpha 1-6 glycosidic bonds. In particular, mutant JPG2 has an enzyme activity of 1.2 times that of the wild type for alpha 1-3 and 34.5 times that of the wild type for alpha 1-6 glycosidic bond. When various bond type glycosidic bonds are cut off for complex glycoprotein, only one mannosidase of the invention is needed to be added, and continuous digestion is not needed to be carried out by adding alpha 1-3 mannosidase or/and alpha 1-6 mannosidase. Therefore, the mannosidase mutant can simplify the sugar chain detection step of glycoprotein and shorten the detection time.

Description

Mannosidase mutant and recombinant gene thereof

Technical Field

The invention relates to the field of bioengineering, in particular to a mannosidase mutant and a recombinant gene thereof.

Background

Glycosylation plays a critical role in the function and stability of proteins, especially in the biomedical field. N-glycosylation, a widely occurring post-translational modification, involves the attachment of sugar chains to amino acid residues of proteins. The structures of these sugar chains are complex and diverse, including complex, hybrid and high mannose-type structures, and they play important roles in protein folding, stability, and recognition and signaling between cells. Mannose, a key component in the structure of the sugar chain, is linked in three different forms (α1-2, α1-3, α1-6) by an α glycosidic bond, affecting the final structure and function of the sugar chain.

In the development and optimization of biotherapeutic drugs, it is important to know and control the glycosylation state, especially the structure and distribution of mannose. The glycosylation pattern of a drug can significantly affect its pharmacokinetic properties, potency, and safety, e.g., affect the half-life, immunogenicity, and biological activity of the drug. Alpha-mannosidase is a key enzyme that helps analyze and characterize the glycosylation structure of proteins by specifically hydrolyzing alpha-mannose residues on the sugar chain, thereby providing a thorough understanding of protein function.

Although α -1,2,3,6 mannosidases are active on all of the α 1-2, α1-3 and α1-6 mannosidic bonds, they have differences in cleavage efficiency for different types of mannosidic bonds, with the cleavage efficiency for α1-2 being the highest and the cleavage efficiency for α1-6 being the lowest. This difference in efficiency is particularly pronounced when dealing with complex sugar chain structures, and the α1-2,3,6 mannosidase requires more than 24 hours of incubation at 37 ℃ to completely digest mannose with complex sugar chain structures. This limitation affects the efficiency and accuracy of protein characterization, particularly in the development and optimization of biotherapeutic drugs. Therefore, how to improve the cleavage efficiency of alpha 1-2,3,6 mannosidase on alpha 1-6 is a technical problem to be solved.

Disclosure of Invention

In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide a mutant with higher enzymatic activity for alpha 1-6 than wild type alpha 1-2,3,6 mannosidase.

In order to achieve the above purpose, the present invention provides the following technical solutions: a mannosidase mutant has a wild type sequence shown in SEQ ID NO. 1. The wild-type mannosidase sequence is derived from a gene library Uniport: o18497.

A mutant of mannosidase takes wild mannosidase as a female parent and carries out the following sequence transformation:

1) Deleting 1-70 amino acids;

2) The following sets of mutations were made: a827v+l829e+q950r+w952Y;

The amino acid position is referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

Preferably, a mutant of mannosidase, using wild-type mannosidase as the female parent according to SEQ ID NO:1, is modified in sequence to include the following mutation sets in addition to the modifications described in 1) +2): 3) S488Y+G501 A+E504L/R+K510E+A559I, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

Preferably, a mannosidase mutant takes the wild mannosidase shown in SEQ ID NO. 1 as a female parent, and the sequence modification is performed by the sequence modification except 1) +2) +3), and the mutant also comprises the following mutation sets: R492K + S503A + T563A, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

Preferably, a mannosidase mutant takes the wild mannosidase shown in SEQ ID NO. 1 as a female parent, and the sequence modification is performed by the sequence modification except 1) +2) +3), and the mutant also comprises the following mutation sets: i491l+r492k+s503a+t563A, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

Preferably, a mannosidase mutant takes the wild mannosidase shown in SEQ ID NO. 1 as a female parent, and the sequence modification is performed by the sequence modification except 1) +2) +3), and the mutant also comprises the following mutation sets: R492K, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

Preferably, a mannosidase mutant takes the wild mannosidase shown in SEQ ID NO. 1 as a female parent, and the sequence modification is performed by the sequence modification except 1) +2) +3), and the mutant also comprises the following mutation sets: i491l+s503a+t563A, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

The second object of the present invention is to provide a genetic material capable of expressing the mannosidase mutant.

In order to achieve the above purpose, the present invention provides the following technical solutions:

a mannosidase recombinant gene capable of expressing the DNA or RNA of the above mannosidase mutant.

The present invention also provides a recombinant plasmid capable of expressing the mannosidase mutant.

a mannosidase recombinant plasmid comprising the above mannosidase recombinant gene.

The fourth object of the present invention is to provide a recombinant cell capable of expressing the above mannosidase mutant.

In order to achieve the above purpose, the present invention provides the following technical solutions: a mannosidase recombinant cell comprising DNA or RNA capable of expressing the above mannosidase mutant.

Compared with the prior art, the invention has the advantages that: the mannosidase mutant has better enzyme digestion activity on alpha 1-2, alpha 1-3 and alpha 1-6. When various bond type glycosidic bonds are cut off for complex glycoprotein, only one mannosidase of the invention is needed to be added, and continuous digestion is not needed to be carried out by adding alpha 1-3 mannosidase or/and alpha 1-6 mannosidase. Therefore, the mannosidase mutant can simplify the sugar chain detection step of glycoprotein and shorten the detection time.

Detailed Description

The term "recombinant gene" refers to DNA or RNA capable of expressing the mannosidase or mannosidase mutant according to the invention. Typically, recombinant genes are initially synthesized in vitro by solid phase phosphoramidite triester or TdT biosynthesis or other suitable techniques known in the art. Amplification may be performed by PCR or other suitable techniques known in the art, with the template sequence. With the recombinant strain, the strain can be further amplified on a large scale by means of culturing the strain. In certain embodiments, the recombinant gene may further include a cleavage site residual sequence, other accessory elements such as control elements (e.g., promoters, etc.), labeling substances (e.g., fluorescent markers, etc.), and other sequences that do not affect expression of the gene of interest.

The term "cloning scar" refers to a promoter sequence whose protein expression depends on initiating transcription of messenger ribonucleic acid (mRNA), followed by a ribosome-binding site (RBS) that attracts the translation machinery, followed by a signal peptide sequence that facilitates protein transport to the periplasm. The mature protein is typically cloned after the signal peptide, and the mature protein is cleaved from the signal peptide by the signal peptidase as it passes through the membrane. However, when cloning a construct after a signal peptide, a restriction enzyme typically requires a specific sequence to cleave the DNA, which leaves a cloning scar after the signal peptide sequence.

The term "signal peptide" refers to a short peptide (typically 16-30 amino acids long) present at the N-terminus of most newly synthesized proteins that are intended to enter the secretory pathway. It may also be referred to as a signal sequence, targeting signal, localization sequence, transit peptide, leader sequence or leader peptide. The signal peptide is typically cleaved from the protein by a signal peptidase.

The term "signal peptide cleavage site" refers to a dipeptide between which a signal peptidase cleaves a signal peptide from a mature protein. In most, but not all cases, the dipeptide is Ala-Ala. The signal peptide cleavage site can be calculated using an algorithm such as SignalP 4.1, which is described in http:

//www.cbs.dtu.dk/services/SignalP/(Center for Biological Sequence Analysis,Technical University of Denmark) Can be used on line.

The term "promoter" refers to a region of DNA that initiates transcription of a particular gene (written to mRNA). Promoters are usually located near the transcription initiation site of a gene, on the same strand and upstream of the DNA (pointing to the 5' region of the sense strand). The promoter may be inducible, meaning that expression of the gene operably linked to the promoter may be turned on by the presence of an inducer substance. Alternatively, the promoter may be constitutive, i.e., it is not regulated by any inducer substance.

The abbreviation "RBS" refers to a ribosome-binding site, or a binding site for a ribosome. This is the sequence of nucleotides upstream of the start codon of the mRNA transcript, which is responsible for the recruitment of ribosomes during the initiation of protein translation.

The term "expression" refers to the process by which DNA is transcribed into messenger RNA (mRNA) and then translated into protein.

The term "expression vector" has the ability to incorporate and express heterologous polynucleic acid fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The choice of an appropriate expression vector is within the knowledge of the skilled person.

The term "chassis cell" refers to a suitable host vector for expression comprising the DNA of the invention. The host may comprise any organism capable of comprising and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. The chassis cell may be a prokaryote or eukaryote, single or multiple cells including mammalian cells, plant cells, fungi, etc. According to the prior art, the person skilled in the art can realize the heterologous expression of the recombinant DNA of the invention in different published chassis cells by adjusting parameters for a limited number of experiments. The chassis cell is selected from at least one of Escherichia coli, pichia pastoris, saccharomyces cerevisiae, hansenula, candida, rhodotorula, bacillus, escherichia, salmonella, clostridium, streptomyces, staphylococcus, neisseria, and Shigella. The present invention is merely exemplified by the types of chassis cells, and is not limited to the types of chassis cells. The chassis cells are preferably E.coli, suitable E.coli strains (including many others) include BL21(DE3),C600,DH5αF′,1113101,JM83,JM101,JM103,JM105,JM107,JM109,JM110,MC1061,MC4100,MM294,NM522,NM554,TGI,χ1776,XL1-Blue and Y1089 ⁺, and the like. The above E.coli strains are all commercially available strains.

The term "identity" means that the residues in the two sequences are identical when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms such as those described herein. For example, two sequences are said to have 50% identity if, when properly aligned, the corresponding fragments of the two sequences have identical residues at 5 of the 10 positions. Most bioinformatic programs report percent identity of aligned sequence regions, which are typically not the entire molecule. If the alignment is long enough and contains enough identical residues, then the expected value can be calculated, indicating that the same level in alignment is unlikely to occur randomly.

The present invention will be described in further detail with reference to examples.

Example 1

Proteins are the material basis of life and are important components of human cells and tissues. All important components in the human body require the participation of proteins, which play a very important role in the vital activities of cells and organisms. It can be said that there is no life without protein. The protein in human body has very various kinds and different functions, and some of the protein can form human tissues, some of the protein can provide energy, some of the protein can participate in metabolism and transportation of substances, some of the protein can promote growth and development, and some of the protein can regulate immune function. Different proteins take on different roles and roles, the function of which is determined by the structure of the protein. The 3D structure of the protein is determined by the amino acid sequence of the protein. Therefore, the design of proteins depends on the correspondence between the structure and the sequence, and it is necessary to design proteins having specific functions, and it is necessary to design sequences conforming to the functional structures. Knowing and designing proteins is of great importance to drive innovative advances in biology and medicine.

It is a very difficult task to design a protein sequence for a specific function, and what structure and function the designed sequence finally assumes is unexpected. And the sample space for a fixed length protein sequence is also quite large. In order to accomplish the above work, the literature developed a protein design platform based on deep learning algorithms, lesign. The platform realizes the functions of protein structure prediction, sequence design, result evaluation and the like. The functional modules cooperate through interfaces to form a calculation pipeline integrating prediction, design and evaluation.

And (3) carrying out sequence design on the wild mannosidase (the amino acid sequence is shown as SEQ ID NO. 1) by using a Lesign platform, and finally obtaining the optimal enzyme variant on the calculation level.

Construction of recombinant cells:

The nucleotide sequences of the genes of interest were all synthesized by Beijing qing Biotechnology Co., ltd, and these nucleotide sequences were inserted into expression vectors. Specifically, it was inserted into plasmid pET28a (+) to obtain the corresponding plasmid. The synthesized plasmids were then transferred into chassis cells (e.coli BL21 (DE 3)), whereby e.coli strains containing different plasmids were constructed. There are many other plasmids and chassis cells available in the art, and only one specific protocol is provided herein.

Expression and purification of mannosidase:

3. Mu.L of recombinant bacteria were streaked on LB solid medium and placed in an incubator at 37℃overnight for cultivation. Single colonies cultured overnight were picked and inoculated into fresh LB medium or M9 medium (containing 20. Mu.g/mL kanamycin), shake cultured overnight at 37℃with shaking at 200rpm, added with isopropyl thiogalactoside (IPTG) at a final concentration of 1mM, and induced to express for 24h at 37℃with 200 rpm. There are many other methods available in the art for inducing expression of recombinant bacteria, and only one specific protocol is provided herein.

Cells were collected by centrifugation and cells were resuspended in lysis buffer. Ultrasonic crushing, separating supernatant and precipitate, and purifying to obtain mannosidase. According to the prior art, the purification of mannosidase can be achieved by a person skilled in the art by adjusting the parameters with a limited number of experiments, which are not described in detail here.

Standard curve: free mannose solutions of different concentrations were prepared, then PBS buffer and developing solution (glucose oxidase (100U/mL), horseradish peroxidase (10U/mL) and dianisidine dihydrochloride (0.7 mg/mL) were added to the mannose solution) were added each time, deionized water was finally added to a volume of 1000. Mu.L, pH 7.4, and the mannose concentration in each reaction system was 20mM,40mM,60mM,80mM,100mM,120mM,140mM,160mM,180mM,200mM, respectively. After 3 hours of reaction, the absorbance at A ₄₅₀ was measured, and a corresponding standard curve was prepared to obtain a standard curve.

The enzyme activity determination method comprises the following steps:

1. referring to the table below, the reaction mixture (for example 1000. Mu.L) was placed in an ice bath.

2. The reaction system is evenly mixed, and then liquid is gathered at the bottom of the tube by adopting a low-speed centrifugation mode.

3. The reaction system was incubated at 37℃for 3 hours.

4. The reaction system after incubation was inactivated at 75℃for 10 minutes.

5. The absorbance at A ₄₅₀ was measured.

6. And inputting the measured absorbance into a standard curve, calculating to obtain the powerful mannose concentration, and then calculating to obtain the enzyme activity of mannosidase.

The unit U of enzyme activity is: in a reaction system of PBS buffer solution, pH 7.4 and 37 ℃, the enzyme amount of the mann-alpha-1, 2-Manalpha-OCH ₃ releasing 1nmol of mannose per hour is hydrolyzed. Specific enzyme activity U/. Mu.g: enzyme activity units contained per μg of enzyme protein.

The following point mutations were performed using wild-type mannosidase (WT, amino acid sequence shown in SEQ ID NO. 1) as a parent. And the enzyme activities of the wild type and the mutant to disaccharide Man-alpha-1, 2-Man-alpha-OCH ₃ are measured according to the above enzyme activity measurement method, and specific values are shown in Table 1.

TABLE 1

As is clear from Table 1, the wild-type mannosidase has a higher enzyme activity on the. Alpha.1-2 glycosidic bond, and the enzyme activity of the mannosidase mutant of the present invention on the. Alpha.1-2 glycosidic bond is substantially the same as that of the wild-type.

The enzyme activity determination method comprises the following steps:

3. The reaction system was incubated at 37℃for 3 hours.

4. The reaction system after incubation was inactivated at 75℃for 10 minutes.

5. The absorbance at A ₄₅₀ was measured.

The unit U of enzyme activity is: in a reaction system of PBS buffer solution, pH 7.4 and 37 ℃, the enzyme amount of the mann-alpha-1, 3-Manalpha-OCH ₃ releasing 1nmol of mannose per hour is hydrolyzed. Specific enzyme activity U/. Mu.g: enzyme activity units contained per μg of enzyme protein.

The following point mutations were performed using wild-type mannosidase (WT, amino acid sequence shown in SEQ ID NO. 1) as a parent. And the enzyme activities of the wild type and the mutant to disaccharide Man-alpha-1, 3-Man-alpha-OCH ₃ are measured according to the above enzyme activity measurement method, and specific values are shown in Table 2.

TABLE 2

As is clear from Table 2, the wild-type mannosidase has lower enzyme activity on the alpha 1-3 glycosidic bond, and the mannosidase mutant of the invention has significantly improved enzyme activity on the alpha 1-3 glycosidic bond. In particular, the enzyme activity of mutant JPG2 is improved to 1.2 times of that of the wild type.

The enzyme activity determination method comprises the following steps:

3. The reaction system was incubated at 37℃for 3 hours.

4. The reaction system after incubation was inactivated at 75℃for 10 minutes.

5. The absorbance at A ₄₅₀ was measured.

The unit U of enzyme activity is: in a reaction system of PBS buffer solution, pH 7.4 and 37 ℃, the enzyme amount of the mann-alpha-1, 6-Manalpha-OCH ₃ releasing 1nmol of mannose per hour is hydrolyzed. Specific enzyme activity U/. Mu.g: enzyme activity units contained per μg of enzyme protein.

The following point mutations were performed using wild-type mannosidase (WT, amino acid sequence shown in SEQ ID NO. 1) as a parent. And the enzyme activities of the wild type and the mutant to disaccharide Man-alpha-1, 6-Man-alpha-OCH 3 are measured according to the enzyme activity measuring method, and specific values are shown in Table 3.

TABLE 3 Table 3

As can be seen from Table 3, the wild-type mannosidase has very low enzymatic activity towards the alpha 1-6 glycosidic bond, whereas the mannosidase mutants of the invention show very significant improvement of the enzymatic activity towards the alpha 1-6 glycosidic bond. In particular, mutant JPG2 has 34.5 times the enzymatic activity of the alpha 1-6 glycosidic bond compared with the wild type.

In conclusion, the mannosidase mutant has better digestion activity on alpha 1-2, alpha 1-3 and alpha 1-6. When various bond type glycosidic bonds are cut off for complex glycoprotein, only one mannosidase of the invention is needed to be added, and continuous digestion is not needed to be carried out by adding alpha 1-3 mannosidase or/and alpha 1-6 mannosidase. Therefore, the mannosidase mutant can simplify the sugar chain detection step of glycoprotein and shorten the detection time.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention can be made by one of ordinary skill in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims

1. A mannosidase mutant is characterized in that the mannosidase mutant takes the wild mannosidase as a female parent according to SEQ ID NO. 1, and the following sequence modification is carried out:

1) Deleting 1-70 amino acids;

2) The following sets of mutations were made: a827v+l829e+q950r+w952Y;

2. A mutant mannosidase characterized by: the mannosidase takes the wild-type mannosidase as a female parent, and the sequence modification comprises the sequence modification as claimed in claim 1 and also comprises the following mutation sets: s488Y+G501 A+E504L/R+K510E+A559I, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

3. A mutant mannosidase characterized by: the mannosidase takes the wild-type mannosidase as a female parent, and the sequence modification comprises the sequence modification as claimed in claim 2 and also comprises the following mutation sets: R492K + S503A + T563A, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

4. A mutant mannosidase characterized by: the mannosidase takes the wild-type mannosidase as a female parent, and the sequence modification comprises the sequence modification as claimed in claim 2 and also comprises the following mutation sets: i491l+r492k+s503a+t563A, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

5. A mutant mannosidase characterized by: the mannosidase takes the wild-type mannosidase as a female parent, and the sequence modification comprises the sequence modification as claimed in claim 2 and also comprises the following mutation sets: R492K, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

6. A mutant mannosidase characterized by: the mannosidase takes the wild-type mannosidase as a female parent, and the sequence modification comprises the sequence modification as claimed in claim 2 and also comprises the following mutation sets: i491l+s503a+t563A, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

7. A mutant mannosidase characterized by: the mannosidase takes the wild-type mannosidase as a female parent, and the sequence modification comprises the sequence modification as claimed in claim 2 and also comprises the following mutation sets: R492K + S503A + T563A, said amino acid position being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type mannosidase shown in 1.

8. Recombinant genetic material of mannosidase, characterized in that it is capable of expressing a DNA or RNA of the mannosidase mutant according to any of claims 1-7.

9. A recombinant plasmid of mannosidase, characterized by comprising the recombinant genetic material of mannosidase according to claim 8.

10.A recombinant cell of mannosidase, characterized by comprising a recombinant plasmid of mannosidase according to claim 9.