CN112646800A

CN112646800A - Method for producing immobilized enzyme or immobilized protein, and immobilized enzyme or immobilized protein

Info

Publication number: CN112646800A
Application number: CN201910962047.9A
Authority: CN
Inventors: 罗晖; 常雁红; 田珺玮; 贾瑞琦; 孙宏旭; 汪月
Original assignee: University of Science and Technology Beijing USTB
Current assignee: University of Science and Technology Beijing USTB
Priority date: 2019-10-11
Filing date: 2019-10-11
Publication date: 2021-04-13

Abstract

The invention discloses a method for preparing immobilized enzyme or immobilized protein and the immobilized enzyme or immobilized protein. The method comprises the following steps: covalently binding SpyCatcher to the carrier; constructing target enzyme or target protein fused with SpyTag at the N end or the C end; the vector to which SpyCatcher is covalently bound and the target enzyme or target protein to which SpyTag is fused are brought into contact with each other. The immobilized enzyme or immobilized protein comprises a carrier, a SpyCatcher covalently bound to the carrier, and a target enzyme or target protein having a SpyTag fused to the N-or C-terminus, wherein the target enzyme or target protein is immobilized on the carrier by the SpyTag covalently bound to the SpyCatcher. The invention also relates to a carrier covalently bonded with the Spycatcher and application thereof in selectively immobilizing target enzyme or target protein fused with SpyTag at the N end or the C end from a protein mixture.

Description

Method for producing immobilized enzyme or immobilized protein, and immobilized enzyme or immobilized protein

Technical Field

The present invention relates to a method for producing an immobilized enzyme or an immobilized protein, and more particularly, to a method for producing an immobilized enzyme or an immobilized protein by purifying a target enzyme or a target protein fused with SpyTag from a protein mixture using a carrier to which SpyCatcher is bound and immobilizing the same on the carrier, and an immobilized enzyme or an immobilized protein obtained by the method. The invention also relates to a carrier covalently bonded with the Spycatcher and application thereof in selectively immobilizing target enzyme or target protein fused with SpyTag at the N end or the C end from a protein mixture.

Technical Field

The enzyme (protein) as the biocatalyst has the advantages of mild reaction conditions, high selectivity, high specificity, high conversion rate and the like, so the enzyme (protein) has good industrial application prospect. However, the free enzyme has many limitations in industrial application because its properties are far from industrial requirements. In addition, the target enzyme is often produced along with other contaminating proteins during the manufacturing process, which often require purification methods to remove, and additional purification steps necessarily add cost. On the other hand, in order to meet the demand for large-scale use in industrial applications and to improve the efficiency of separation of the enzyme from the reaction liquid, immobilized enzymes are often used in industrial applications.

Generally, purification of the enzyme and immobilization of the enzyme are two very important links in industrial application of the enzyme. In order to reduce the number of process steps, increase efficiency and reduce costs, attempts have been made to purify and immobilize enzymes in one step, for example, immobilized metal affinity chromatography methods are reported in Gaberc-Porekar V, Menart V, Gaberc-Porekar V, et al.perspectives of immobilized-metal affinity chromatography [ J ]. Jbiochemimphiphysiophysiology methods.2001,49: 335-.

Disclosure of Invention

In order to solve the above problems, the present invention provides a simple process for producing an immobilized enzyme or immobilized protein, which is mild in conditions and stable in binding of an enzyme to a carrier, by separating a target enzyme or target protein from a hetero protein and binding the target enzyme or target protein to the carrier in one step to obtain the immobilized enzyme or immobilized protein. The obtained immobilized enzyme or immobilized protein has high stability, maintains high enzyme activity and can meet the requirement of industrial application. The present invention also provides a vector to which SpyCatcher is covalently bound, and relates to use of the vector to selectively immobilize a target enzyme or a target protein, in which SpyTag is fused to the N-terminus or C-terminus of a protein mixture.

The above problems are solved by the following technical solutions.

1. A method for preparing an immobilized enzyme or an immobilized protein, the method comprising:

-covalently binding SpyCatcher to the carrier;

-constructing a target enzyme or target protein fused at the N-or C-terminus with SpyTag;

contacting the vector to which the SpyCatcher is covalently bound with the target enzyme or the target protein to which the SpyTag is fused.

2. The method of clause 1, wherein the SpyCatcher has the gene coding sequence of SEQ ID No.1, or has the gene coding sequence of SEQ ID No.3, or has the gene coding sequence of SEQ ID No.6, or has the gene coding sequence of SEQ ID No. 7.

3. The method of clauses 1 or 2, wherein the SpyTag has the gene coding sequence of SEQ ID No. 2.

4. The method of any one of items 1 to 3, wherein the carrier is an epoxy-based carrier or an acetaldehyde agarose carrier.

5. The method of any one of items 1 to 4, wherein the target enzyme is L-phenylserine aldolase (LPA) or Leucine Dehydrogenase (LDH), and the target protein is Green Fluorescent Protein (GFP), preferably, the L-phenylserine aldolase has the gene-coding sequence of SEQ ID No.8, and the leucine dehydrogenase has the gene-coding sequence of SEQ ID No. 9.

6. An immobilized enzyme or an immobilized protein obtained by the method of any one of items 1 to 5.

7. An immobilized enzyme or an immobilized protein comprising a carrier, a SpyCatcher covalently bound to the carrier, and a target enzyme or a target protein having a SpyTag fused to an N-terminus or a C-terminus, wherein the target enzyme or the target protein is immobilized on the carrier by the SpyCatcher being covalently bound to the SpyCatcher.

8. The immobilized enzyme or immobilized protein of item 7, wherein the SpyCatcher has the gene coding sequence of SEQ ID No.1, or has the gene coding sequence of SEQ ID No.3, or has the gene coding sequence of SEQ ID No.6, or has the gene coding sequence of SEQ ID No. 7.

9. The immobilized enzyme or immobilized protein of item 7 or 8, wherein the SpyTag has the gene coding sequence of SEQ ID No. 2.

10. The immobilized enzyme or immobilized protein of any one of items 7 to 9, wherein the carrier is an epoxy-based carrier or an acetaldehyde agarose carrier.

11. The immobilized enzyme or protein of any one of items 7 to 10, wherein the target enzyme is L-phenylserine aldolase or leucine dehydrogenase, and the target protein is green fluorescent protein, preferably, the L-phenylserine aldolase has the gene coding sequence of SEQ ID No.8, and the leucine dehydrogenase has the gene coding sequence of SEQ ID No. 9.

12. A vector to which SpyCatcher is covalently bound.

13. The vector of item 12, wherein the SpyCatcher has the gene coding sequence of SEQ ID No.1, or has the gene coding sequence of SEQ ID No.3, or has the gene coding sequence of SEQ ID No.6, or has the gene coding sequence of SEQ ID No. 7.

14. The vector of item 12 or 13, wherein the vector is an epoxy-based vector or an acetaldehyde agarose vector.

15. Use of a vector to which SpyCatcher is covalently bound for selectively immobilizing a target enzyme or a target protein having SpyTag fused at the N-terminus or C-terminus from a protein mixture.

16. The use of clause 15, wherein the SpyCatcher has the gene coding sequence of SEQ ID No.1, or has the gene coding sequence of SEQ ID No.3, or has the gene coding sequence of SEQ ID No.6, or has the gene coding sequence of SEQ ID No. 7.

17. The use of clauses 15 or 16, wherein the SpyTag has the gene coding sequence of SEQ ID No. 2.

18. The use of any one of items 15-17, wherein the carrier is an epoxy-based carrier or an acetaldehyde agarose carrier.

19. The use according to any one of items 15 to 18, wherein the enzyme of interest is an L-phenylserine aldolase or a leucine dehydrogenase, and the protein of interest is a green fluorescent protein, preferably the L-phenylserine aldolase has the gene-coding sequence of SEQ ID No.8, and the leucine dehydrogenase has the gene-coding sequence of SEQ ID No. 9.

The preparation method of the immobilized enzyme or the immobilized protein has the advantages that the target enzyme or the target protein can be separated from the protein mixed solution and immobilized in one step, the immobilization rate is high, the obtained immobilized enzyme keeps high enzyme activity and good thermal stability, and the obtained immobilized protein has good thermal stability.

Drawings

FIG. 1 schematically shows a process for producing an immobilized enzyme or an immobilized protein using acetaldehyde agarose as a carrier.

Detailed Description

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, to the same extent as if each was specifically and individually indicated to be incorporated by reference in its entirety.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

All percentages, parts, ratios, etc., are by weight unless otherwise indicated.

When an amount, concentration, or other value or parameter is expressed in terms of a range, preferred range, or upper preferable numerical value and lower preferable numerical value, it is understood that any range defined by any pair of upper range limits or preferred numerical values in combination with any lower range limits or preferred numerical values is specifically disclosed, regardless of whether the range is specifically disclosed. Unless otherwise indicated, numerical ranges set forth herein are intended to include the endpoints of the ranges, and all integers and fractions within the ranges.

Herein, the term "formed of … …" or "consisting of … …" is equivalent to "comprising/including". As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or any other variation thereof, are intended to cover a non-exclusive inclusion.

In addition, the word "a" or "an" preceding a certain element or component of the invention is not limiting as to the number of such elements or components. Thus, "a" or "an" should be understood to include one or at least one and the singular forms of a stated element or component also include the plural unless the singular is explicitly stated.

The materials, methods, and examples of the present invention are illustrative only and not intended to be limiting unless otherwise specified. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

When the term "about" is used to describe a value or an endpoint of a range, it should be understood to include within ± 10%, ± 5%, ± 3% or ± 1% of the specific value or endpoint involved. In the present invention, each specific numerical value referred to should be considered modified by "about".

In one aspect of the present application, there is provided a method for preparing an immobilized enzyme or an immobilized protein, the method comprising:

-covalently binding SpyCatcher to the carrier;

In the present application, SpyCatcher and SpyTag have meanings conventional in the art. SpyTag is a polypeptide fragment and SpyCatcher is a protein corresponding thereto, which can form an isopeptide bond covalently (Zakiri BF, J.O.; Celik, E.; Chittock, E.C.; Schwarz-Link, U.S.; Moy, V.T.; Howarth, M.peptide tag formation a rapid covalent bond to a protein, through engineering a bacterial attachment. proceedings of the National Academy of Sciences 2012; 2012,109 (12); E690-E7.).

Preferably, in the present application, SpyCatcher has the gene coding sequence of SEQ ID No.1, or has the gene coding sequence of SEQ ID No.3, or has the gene coding sequence of SEQ ID No.6, or has the gene coding sequence of SEQ ID No. 7.

In the present application, for the sake of convenience of description, SpyCatcher having the gene coding sequence of SEQ ID No.1 is referred to as oSpyCatcher, SpyCatcher having the gene coding sequence of SEQ ID No.6 is referred to as appycatcher, SpyCatcher having the gene coding sequence of SEQ ID No.7 is referred to as cSpyCatcher, and SpyCatcher having the gene coding sequence of SEQ ID No.3 is referred to as spmspycatcher, respectively. It is to be understood that the aforementioned oSpyCatcher, apsycatcher, cSpyCatcher, mSpyCatcher are all encompassed within the term "spycatccher".

The SpyCatcher having the gene coding sequence of SEQ ID No.3, SEQ ID No.6 or SEQ ID No.7 is a mutant of SpyCatcher having the gene coding sequence of SEQ ID No. 1. Wherein the SpyCatcher having the gene coding sequence of SEQ ID No.6 is a mutant obtained by adding a plurality of lysine peptide chains (LysGlyLysGlyLysGly) to the C-terminus of the protein, based on the SpyCatcher having the gene coding sequence of SEQ ID No. 1; the Spycatcher with the gene coding sequence of SEQ ID No.7 is a mutant obtained by mutating lysine at 28, 37 and 64 positions to arginine on the basis of the Spycatcher with the gene coding sequence of SEQ ID No. 1; the SpyCatcher with the gene coding sequence of SEQ ID No.3 is a mutant combining the two mutation modes on the basis of the SpyCatcher with the gene coding sequence of SEQ ID No. 1.

In the method of the present invention, the method of covalently binding SpyCatcher to the carrier is an enzyme (protein) immobilization method which is conventional in the art. The immobilization method herein is carried out by binding an amino group in SpyCatcher to a group capable of forming a covalent bond with an amino group on the carrier.

In the present invention, the vector combined with aspycutcher, cSpyCatcher, and mSpyCatcher can further improve the immobilization rate and immobilization stability of the target enzyme or the target protein, relative to the vector combined with ospycutcher. Without wishing to be bound by theory, the inventors believe that the addition of a peptide chain containing multiple lysines (LysGlyLysGlyLysGly gly) to the C-terminus of oSpyCatcher (appycatcher) enables directional immobilization and more secure binding of the peptide chain to a vector, and the mutation of lysines at positions 28, 37 and 64 of oSpyCatcher to arginine (cSpyCatcher) enables the immobilization direction of SpyCatcher on the vector to be more favorable for subsequent binding of SpyTag, so that the mSpyCatcher combining the two mutation modes has both advantages.

Generally, when the carrier is an agarose carrier, the agarose carrier is first pretreated with epoxypropanol and sodium periodate to have aldehyde groups, and then bound by covalent bonds between amino groups in SpyCatcher and aldehyde groups on the carrier. When the carrier is an epoxy carrier, the carrier is usually bonded directly by covalent bonding of an amino group in SpyCatcher to an epoxy group on the carrier without pretreatment of the carrier. When other conventional carriers in the art are used, the SpyCatcher is covalently bound to the carrier by processing or binding methods known to those skilled in the art.

Preferably, the SpyTag in the present application has the gene coding sequence of SEQ ID No. 2.

In the present application, the SpyTag may be fused to the N-terminus or C-terminus of the target enzyme or target protein. The gene encoding the fusion protein can be obtained by fusing the SpyTag peptide fragment to the N-terminus or C-terminus of the target enzyme or target protein by methods conventional in the art, such as the Megawhop method.

In the present application, the vector to which the spytcher is covalently bound and the target enzyme or the target protein to which the SpyTag is fused are contacted with each other under conditions of, for example, normal temperature and pressure, for example, 4 ℃ to 37 ℃ (for example, 20 ℃ to 35 ℃), for example, 8 to 20 hours, for example, 10 to 16 hours, and the pH is not particularly limited as long as conditions suitable for the presence of the protein without denaturation are satisfied, for example, pH5 to 10, and pH5.5 to 9.

In a further aspect the present application relates to an immobilized enzyme or an immobilized protein obtained according to the process of the invention.

In yet another aspect the present application relates to an immobilized enzyme or an immobilized protein comprising

-a carrier;

-SpyCatcher covalently bound to the carrier; and

a target enzyme or a target protein having a SpyTag fused to the N-terminus or C-terminus,

wherein the target enzyme or the target protein is immobilized on the carrier by covalently binding the SpyTag to the SpyCatcher.

The foregoing descriptions and definitions of the processes for producing immobilized enzymes or immobilized proteins are also applicable to the immobilized enzymes or immobilized proteins of the present aspect.

In a further aspect the present application relates to a carrier to which a SpyCatcher is covalently bound, and what has been said above for covalently binding SpyCatcher to the carrier in the method for preparing an immobilized enzyme or an immobilized protein is also applicable in this respect.

In a further aspect of the present application, which relates to the use of a carrier covalently bound to SpyCatcher for selectively immobilizing a target enzyme or a target protein having SpyTag fused at the N-terminus or C-terminus from a protein mixture, the foregoing description and definitions of the method for producing an immobilized enzyme or immobilized protein are also applicable to the use of the present aspect.

Examples

The present invention will be described in more detail below by way of examples, but it should be understood that the scope of the present invention is not limited to the examples.

The measurement and calculation methods used in the examples:

1) thermostability of the enzyme

Free or immobilized enzyme was incubated at 60 ℃ and samples were taken at regular intervals to determine residual activity.

2) Immobilization Rate of Green Fluorescent Protein (GFP)

Fluorescence intensities of free GFP and immobilized GFP in 100mM Phosphate Buffer (PB), at 28 ℃ and pH8.5 were measured using a multifunctional microplate reader at excitation and emission wavelengths of 488nm and 508nm, respectively.

The method for calculating the immobilization rate of the SpyTag-GFP comprises the following steps:

the ratio of the SpyTag-GFP immobilization (SpyTag-GFP fluorescence in the supernatant at the time of immobilization-SpyTag-GFP residual fluorescence in the supernatant at the end of immobilization)/SpyTag-GFP fluorescence in the supernatant at the time of immobilization x 100%

3) L-Phenylserine Aldolase (LPA) Activity and immobilization Rate

LPA enzyme activity definition: the amount of enzyme required to catalyze the formation of 1. mu. mol of benzaldehyde from L-phenylserine per minute at 30 ℃ at pH8.5 and a L-phenylserine concentration of 10mmol/L was 1 activity unit (U).

The concentration of the generated benzaldehyde is measured at the ultraviolet of 290nm at the temperature of 30 ℃ by using an EnSpire2300 multifunctional microplate reader to calculate the enzyme activity.

The SpyTag-LPA immobilization rate calculation method comprises the following steps:

the immobilization rate of SpyTag-LPA (SpyTag-LPA enzyme activity in the supernatant at the time of immobilization zero time-SpyTag-LPA enzyme activity in the supernatant at the time of immobilization completion)/the enzyme activity of SpyTag-LPA in the supernatant at the time of immobilization zero time is multiplied by 100%

4) Enzyme activity and immobilization Rate of leucine dehydrogenase Activity (LDH)

LDH enzyme activity was defined: with 900mM NH pH 9.5₃-NH₄Preparing a substrate solution containing 4.5mM of 2-ketobutyric acid and 0.204mM of NADH by using a Cl buffer solution, detecting the enzyme activity by using an EnSpire2300 multifunctional microplate reader at 30 ℃, and defining the enzyme quantity required for catalyzing and consuming 1 mu mol of NADH per minute as 1U.

Measuring NADH concentration change value and time at the ultraviolet of 340nm at 30 ℃ by using an EnSpire2300 multifunctional microplate reader to calculate enzyme activity.

The method for calculating the immobilization rate of the SpyTag-LDH comprises the following steps:

the immobilization rate of SpyTag-LDH (SpyTag-LDH enzyme activity in the immobilized zero-time supernatant-SpyTag-LDH enzyme activity in the supernatant at the end of immobilization)/the activity of SpyTag-LDH enzyme in the immobilized zero-time supernatant is multiplied by 100%

Example A: preparation of SpyCatcher-bound vectors

Construction of SpyCatcher gene engineering expression bacteria and culture

Using Megawhop (Sanchs J, Layla Fern & ndez, Carballera J D, et al. improved PCR method for the creation of cultivation mutagenesis of microorganisms in direct evaluation: application to differentiation-to-amplification templates [ J ]. Applied Microbiology and Biotechnology,2008,81(2):387-397.), a pET28a SpyTag-GFP-SpyCatch having the gene coding sequence of SEQ ID No.5 was used as a template to construct pET28 a-photoscatch; adopting primers of DNA sequences of SEQ ID No.14 and SEQ ID No.15, obtaining a first round PCR product through PCR, then using the first round PCR product as a long primer in a second round of whole plasmid amplification to perform PCR to obtain a second round PCR product, performing DpnI enzymolysis on the second round PCR product obtained by Megawhop, then performing DpnI enzymolysis to transform E.coli BL21(DE3) competent cells, and obtaining a genetic engineering strain E.coli BL21(DE3)/pET28a-oSPycatcher through DNA sequencing verification of plasmids.

pET28a-oSpycatcher is used as a template, primers with sequences of SEQ ID No.16 and SEQ ID No.17DNA are adopted, a mutant (aSpycatcher, gene coding sequence SEQ ID No.6) of a gene coding sequence with SEQ ID No.6 and a polylysine peptide chain (LysGlyLysGlyLysGly) added at the C-terminal is constructed by a Megawhop method, and a genetic engineering strain E.coli BL21(DE3)/pET28a-aSpycatcher is finally obtained through sequencing verification.

Similarly, pET28a-oSpyCatcher is used as a template, primers of SEQ ID No.18, SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21DNA sequences are adopted, a mutant (cSpyCatcher, a gene coding sequence SEQ ID No.7) for mutating three lysines (28 th, 37 th and 64 th) into arginine is constructed by a Megawhop method, and finally, a genetic engineering strain E.coli BL21(DE3)/pET28a-cSpyCatcher is obtained.

Finally, combining the two mutation methods to construct a mutant (mSpyCatcher), taking cSpycatcher as a template, adopting primers with sequences of SEQ ID No.16 and SEQ ID No.17DNA, and carrying out PCR by a Megawhop method by using the primers for constructing the aSpyCatcher to obtain the mutant mSpyCatcher (gene coding sequence SEQ ID No.3), wherein the constructed genetically engineered bacterium is E.coli BL21(DE3)/pET28 a-mSpyCatcher.

Genetically engineered Escherichia coli BL21(DE3)/pET28a-oSPycatcher, BL21(DE3)/pET28a-mSPycatcher, BL21(DE3)/pET28a-aSPycatcher, BL21(DE3)/pET28a-cSpycatcher were cultured in LB medium at 37 ℃ and 200r/min for 12 hours, and inoculated in a lactose medium at 1% inoculum size, and cultured at 28 ℃ and 200r/min for 24 hours. The cells were harvested by centrifugation at 4000rpm for 10 min. Wherein the LB culture medium and the lactose culture medium comprise the following components:

LB culture medium: 1% NaCl, 1% peptone, 0.5% yeast powder and 50. mu.g/mL kanamycin.

Lactose self-induction medium: 10g/L of peptone, 5g/L of yeast powder and Na₂HPO₄ 8.95g/L，KH₂PO₄ 3.4g/L，NH₄Cl 2.67g/L，Na₂SO₄ 0.7g/L，MgSO₄0.24g/L, 5g/L of glycerol, 0.5g/L of glucose, 2g/L of lactose and 50 mu g/mL of kanamycin.

Purification of SpyCatcher

The harvested cells were resuspended in 0.1M phosphate buffer (pH8.5) and disrupted by sonication. The conditions of the ultrasound were: the ultrasonic treatment is carried out for 5 seconds at intervals of 5 seconds, 99 cycles are carried out, and the ultrasonic power is 200W, so that the crushing is carried out twice. Centrifuging at 20000rpm for 5min at 4 deg.C to obtain supernatant as protein solution containing SpyCatcher.

The various SpyCatcher protein solutions were loaded onto Ni-NTA resin columns, the pH was adjusted to pH8.5, the columns were washed with 30mM imidazole buffer, contaminating proteins were washed off, and then eluted with 200mM imidazole buffer to obtain various SpyCatcher purified solutions, i.e., oSpyCatcher purified solution, aSpyCatcher purified solution, csspycatcher purified solution, and mspycaptcher purified solution. Storing at 4 deg.C for use.

i) Immobilized on acetaldehyde agarose carrier

At room temperature will wash 6% crosslinked agarose weighing 5.6g, in ice bath slowly adding 1M NaOH and 0.5M NaBH₄The mixture was 4mL, then 1.6mL of epoxypropanol (final concentration 2M) was added and reacted for 15h at 25 ℃ in a shaker at 200 rpm. The stirred agarose was washed and drained. 5g of deionized water (43 mL) was added, and 0.23g of sodium periodate (20 mM final concentration) was added thereto. Shaking at 100rpm for 2h at 25 ℃. The support was washed with copious amounts of deionized water (500mL) and filtered with suction.

1g of the carrier was weighed, and different amounts of the above SpyCatcher purified solution and 0.2M sodium bicarbonate buffer were added to adjust the pH of the system to 10. Shaking at 100rpm for 3h at 25 deg.C, adding sodium borohydride to give a final concentration of 1mg/ml after 3h, stirring at room temperature for 30min, washing with 25mM phosphate buffer (pH8.0), and draining. The oSpyCatcher-agarose vector, the aSpyCatcher-agarose vector, the cSpycatcher-agarose vector and the mSpyCatcher-agarose vector were obtained, respectively. Washing and pumping to dry for later use.

ii) immobilization on an epoxy-based Carrier

1g of the carrier was weighed, and different amounts of the above SpyCatcher purified liquid and 1.25M phosphate buffer were added to adjust the pH of the system to 8. And shaking the mixture at 25 ℃ and 200rpm for 24h to obtain an oSpyCatcher-epoxy vector, an aSpyCatcher-epoxy vector, a cSpyCatcher-epoxy vector and an mSpyCatcher-epoxy vector respectively. Washing and pumping to dry for later use.

Example B: enzyme or protein fused with SpyTagPreparation of

The method of Megawhop is used, the pET28a SpyTag-GFP-SpyCatcher is used as a template to construct pET28a-SpyTag-GFP with SEQ ID No.4, primers with SEQ ID No.12 and SEQ ID No.13DNA sequences are adopted to obtain a first round of PCR product through PCR, and the first round of PCR product is used as a long primer during second round of whole plasmid amplification to perform PCR to obtain a second round of PCR product. And (3) carrying out DpnI enzymolysis on the second round PCR product obtained by Megawhhop, then carrying out enzymolysis on the DpnI to transform E.coli BL21(DE3) competent cells, and carrying out sequencing verification to obtain a genetic engineering strain E.coli BL21(DE3)/pET28 a-SpyTag-GFP. The GFP in pET-SpyTag-GFP was exchanged for L-phenylalanine aldolase (LPA) having the coding sequence of the gene SEQ ID No.8 and Leucine Dehydrogenase (LDH) having the coding sequence of the gene SEQ ID No.9 using the method of GoldenGate (Engler, C., & Marillonnet, S. (2014). gold gate cloning. in DNA cloning and assembly methods (pp.119-131). Humana Press, Totowa, NJ.).

Firstly, a plasmid framework (pET-SpyTag plasmid framework) containing BsaI enzyme cutting sites and target fragments (LPA and LDH) are constructed, primers with SEQ ID No.22 and SEQ ID No.23 are adopted when the plasmid framework is constructed, primers with SEQ ID No.24, SEQ ID No.25, SEQ ID No.26 and SEQ ID No.27 are adopted when the target fragments (LPA and LDH) are constructed, and a GoldenGate method is used for obtaining recombinant plasmids pET-SpyTag-LPA (the gene coding sequence of SpyTag-LPA is SEQ ID No.10) and pET-SpyTag-LDH (the gene coding sequence of SpyTag-LDH is SEQ ID No.11) in an enzyme cutting connection mode.

Genetically engineered Escherichia coli BL21(DE3)/pET28a-SpyTag-GFP, BL21(DE3)/pET28a-SpyTag-LPA or BL21(DE3)/pET-SpyTag-LDH was cultured in LB medium at 37 ℃ and 200r/min for 12 hours, inoculated in a lactose medium at 1% inoculum size, and cultured at 28 ℃ and 200r/min for 24 hours. The cells were harvested by centrifugation at 4000rpm for 10 min. Wherein the LB culture medium and the lactose culture medium comprise the following components:

The harvested cells were resuspended in 0.1M phosphate buffer (pH8.5) and disrupted by sonication. The conditions of the ultrasound were: the ultrasonic treatment is carried out for 5 seconds at intervals of 5 seconds, 99 cycles are carried out, and the ultrasonic power is 200W, so that the crushing is carried out twice. Centrifuging at 20000rpm for 5min at 4 deg.C to obtain supernatant as mixed protein solution containing target protein (SpyTag-GFP, SpyTag-LPA or SpyTag-LDH) fused with SpyTag.

Example C1: immobilization of GFP: oSPyCatcher-agarose vector + SpyTag-GFP

An OspyCatcher-agarose vector was prepared as in example A, and immobilized using 1mg of OspyCatcher per gram of vector.

The resulting oSpyCatcher-agarose vector was mixed with SpyTag-GFP prepared as described in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of oSpyCatcher-agarose vector, and the mixture was placed at 28 ℃ and pH adjusted to 5.5 with HCl.

The supernatant was sampled at mixing zero and 20h after mixing and the fluorescence of the supernatant was measured as described previously. According to the formula, the immobilization rate of SpyTag-GFP on the OspyCatcher-agarose carrier is 40.78% after 20 h.

Example C2: immobilization of GFP: mSpyCatcher-agarose vector + SpyTag-GFP

mSPyCatcher-agarose vectors were prepared as in example A and were immobilized using 1mgmSPyCatcher per gram of vector.

The mSPyCatcher-agarose vector thus prepared was mixed with the SpyTag-GFP prepared in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per g of mSPyCatcher-agarose vector, and the mixture was placed at 28 ℃ and the pH was adjusted to 5.5 with HCl.

The supernatant was sampled at mixing zero and 20h after mixing and the fluorescence of the supernatant was measured as described previously. The immobilization rate of SpyTag-GFP on mspyCatcher-agarose vector over 20h was 55.56% as calculated according to the above formula.

Example C3: immobilization of GFP: oSPyCatcher-agarose vector + SpyTag-GFP

An OspyCatcher-agarose vector was prepared as in example A, and immobilized using 5mg of OspyCatcher per gram of vector.

The resulting oSpyCatcher-agarose vector was mixed with SpyTag-GFP prepared as described in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of oSpyCatcher-agarose vector, and the mixture was placed at 30 ℃ and pH adjusted to 6.5 with HCl.

The supernatant was sampled at mixing zero and 16h after mixing and the fluorescence of the supernatant was measured as described previously. According to the formula, the immobilization rate of the SpyTag-GFP on the OspyCatcher-agarose carrier after 16h is 71.21%.

Example C4: immobilization of GFP: mSpyCatcher-agarose vector + SpyTag-GFP

mSPyCatcher-agarose vectors were prepared as in example A and were immobilized using 5mg mSPyCatcher per gram of vector.

The mSPyCatcher-agarose vector thus prepared was mixed with the SpyTag-GFP prepared in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per g of mSPyCatcher-agarose vector, and the mixture was placed at 30 ℃ and pH was adjusted to 6.5 with HCl.

The supernatant was sampled at mixing zero and 16h after mixing and the fluorescence of the supernatant was measured as described previously. The immobilization rate of SpyTag-GFP on mspyCatcher-agarose vector was 82.90% over 16h, calculated according to the formula above.

Example C5: immobilized LPA: oSPyCatcher-agarose vector + SpyTag-LPA

The resulting oSpyCatcher-agarose vector was mixed with SpyTag-LPA prepared in example B above in a centrifuge tube, 5mg of SpyTag-LPA was added per gram of oSpyCatcher-agarose vector, placed at 25 ℃, and the pH adjusted to 7.5 with HCl.

The supernatant was sampled at mixing zero and 22h after mixing and the enzyme activity of the supernatant was measured as described previously. The immobilization rate of SpyTag-LPA on the OspyCatcher-agarose vector was 59% after 22h, calculated according to the formula above.

Thermal stability:

the thermostability of free LPA enzyme and immobilized SpyTag-LPA in this example was determined according to the assay methods described above.

The enzyme activity of free LPA enzyme at 0 time is 9.50U/mL, the enzyme activity of free LPA enzyme after 100min is 2.67U/mL, and the residual enzyme activity ratio is 28.13%.

The enzyme activity of the immobilized LPA enzyme is 26.96U/g at the time of 0, the enzyme activity of the immobilized LPA enzyme is 16.17U/g after 100min, and the residual enzyme activity ratio is 59.96%.

Example C6: immobilized LPA: mSpyCatcher-agarose vector + SpyTag-LPA

The mSPyCatcher-agarose vector thus prepared was mixed with the SpyTag-LPA prepared in example B above in a centrifuge tube, 5mg of SpyTag-LPA was added per g of mSPyCatcher-agarose vector, and the mixture was placed at 25 ℃ and pH was adjusted to 7.5 with HCl.

The supernatant was sampled at mixing zero and 22h after mixing and the enzyme activity of the supernatant was measured as described previously. The immobilization rate of SpyTag-LPA on mspyCatcher-agarose vector was 73% over 22h, calculated according to the formula above.

Thermal stability:

The enzyme activity of free enzyme LPA at 0 moment is 9.50U/mL, the enzyme activity of free enzyme LPA after 100min is 2.67U/mL, and the residual enzyme activity ratio is 28.13%.

The enzyme activity of the immobilized LPA enzyme is 34.07U/g at 0 moment, the enzyme activity of the immobilized LPA enzyme is 22.84U/g after 100min, and the residual enzyme activity ratio is 67.05%.

Example C7: immobilized LDH: oSPyCatcher-agarose vector + SpyTag-LDH

The resulting oSpyCatcher-agarose vector was mixed with SpyTag-LDH prepared in example B above in a centrifuge tube, 5mg of SpyTag-LDH was added per gram of oSpyCatcher-agarose vector, and the mixture was placed at 20 ℃ and pH 8.5.

The supernatant was sampled at mixing zero and 15h after mixing and the enzyme activity of the supernatant was measured as described previously. The immobilization rate of SpyTag-LDH on the OspyCatcher-agarose vector was 54% after 15h, calculated according to the formula above.

Thermal stability:

the thermal stability of free LDH enzyme and immobilized SpyTag-LDH in this example were each determined according to the assay methods described previously.

The enzyme activity of the free LDH enzyme at the time 0 is 12.72U/mL, the enzyme activity of the free LDH enzyme after 100min is 2.83U/mL, and the ratio of the residual enzyme activity is 22.24%.

The enzyme activity of the immobilized LDH enzyme at 0 moment is 15.92U/g, the enzyme activity of the immobilized LDH enzyme after 100min is 14.68U/g, and the ratio of the residual enzyme activity is 92.19%.

Example C8: immobilized LDH: mSpyCatcher-agarose vector + SpyTag-LDH

The mSPyCatcher-agarose vector prepared was mixed with the SpyTag-LDH prepared in example B above in a centrifuge tube, 5mg of SpyTag-LDH was added per gram of mSPyCatcher-agarose vector, and the mixture was placed at 20 ℃ and pH 8.5.

The supernatant was sampled at mixing zero and 15h after mixing and the enzyme activity of the supernatant was measured as described previously. The immobilization rate of SpyTag-LDH on mspyCatcher-agarose vector over 15h was 85% calculated according to the formula above.

Thermal stability:

The enzyme activity of the free LDH enzyme at the time 0 is 12.72U/mL, the enzyme activity of the free LDH enzyme after 100min is 2.83U/mL, and the ratio of the residual enzyme activity is 22.24%;

the enzyme activity of the immobilized LDH enzyme at 0 time is 23.46U/g, the enzyme activity of the immobilized LDH enzyme is 22.29U/g after 100min, and the ratio of the residual enzyme activity is 95.02%.

Example C9: immobilization of GFP: aPsycatcher-agarose vector + SpyTag-GFP

The aPsycatccher-agarose vector was prepared as in example A, and immobilized using 5mg of aPsycatccher per gram of vector.

The resulting aSpyCatcher-agarose vector was mixed with SpyTag-GFP prepared in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of aSpyCatcher-agarose vector, and the mixture was placed at 28 ℃ and pH was adjusted to 7.5 with HCl.

The supernatant was sampled at mixing zero and 20h after mixing and the fluorescence of the supernatant was measured as described previously. According to the formula, the immobilization rate of SpyTag-GFP on the aPsycatcher-agarose carrier after 20h is 76.78%.

Example C10: immobilization of GFP: cSpyCatcher-agarose vector + SpyTag-GFP

The cSpyCatcher-agarose vector was prepared as in example A, and 5mgcSpyCatcher was used for immobilization per gram of the vector.

The prepared csspycatcher-agarose carrier was mixed with SpyTag-GFP prepared in example B above in a centrifuge tube, and 5mg of SpyTag-GFP was added to each g of the csycatcher-agarose carrier, and the mixture was placed at 28 ℃ and pH was adjusted to 7.5 with HCl.

The supernatant was sampled at mixing zero and 20h after mixing and the fluorescence of the supernatant was measured as described previously. According to the formula, the immobilization rate of SpyTag-GFP on the cSpycatcher-agarose carrier is 77.30% after 20 h.

Example C11: immobilization of GFP: ospycatcher-epoxy vector + SpyTag-GFP

An Ospy catcher-epoxy vector was prepared as in example A, and was immobilized using 20mg of the Ospy catcher per gram of the vector.

The resulting ospycatch-epoxy vector was mixed with SpyTag-GFP prepared as described in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of aspycatch-epoxy vector, and the mixture was placed at 28 ℃ and pH adjusted to 7.5 with HCl.

The supernatant was sampled at mixing zero and 14h after mixing and the fluorescence of the supernatant was measured as described previously. The immobilization rate of SpyTag-GFP on the OspyCatcher-epoxy vector was 50.0% over 14h, calculated according to the formula above.

Example C12: immobilization of GFP: mSpyCatcher-epoxy vector + SpyTag-GFP

A mSPyCatcher-epoxy vector was prepared as in example A and was immobilized using 20mgmSPyCatcher per gram of vector.

The resulting mSPyCatch-epoxy vector was mixed with the SpyTag-GFP prepared in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of mSPyCatch-epoxy vector, and the mixture was placed at 28 ℃ and the pH was adjusted to 7.5 with HCl.

The supernatant was sampled at mixing zero and 14h after mixing and the fluorescence of the supernatant was measured as described previously. The immobilization rate of SpyTag-GFP on the mspyCatcher-epoxy vector over 14h was 72.0% as calculated according to the above formula.

Comparative example 1: blank acetaldehyde agarose vector + SpyTag-GFP

Blank acetaldehyde agarose carrier was prepared as in example A by weighing 1g of activated acetaldehyde agarose carrier, adding deionized water and 0.2M sodium bicarbonate buffer, and adjusting the pH of the system to 10. Shaking at 25 deg.C and 100rpm for 3h to obtain blank acetaldehyde agarose carrier.

The blank acetaldehyde agarose carrier thus obtained was mixed with SpyTag-GFP prepared in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of blank acetaldehyde agarose carrier, and the mixture was placed at 28 ℃ and pH was adjusted to 8.0 with HCl.

The supernatant was sampled at mixing zero and 20h after mixing and the fluorescence of the supernatant was measured as described previously. According to the formula, the immobilization rate of SpyTag-GFP on the blank acetaldehyde agarose carrier is about 0% after 20 h. Meanwhile, the blank acetaldehyde agarose carrier particles after the immobilization treatment have no fluorescence when detected under a fluorescence microscope.

Comparative example 2: blank acetaldehyde agarose Carrier + SpyTag-LPA

Blank acetaldehyde agarose supports were prepared as in comparative example 1.

The blank acetaldehyde agarose carrier thus obtained was mixed with SpyTag-LPA prepared in example B above in a centrifuge tube, 5mg of SpyTag-LPA was added per gram of blank acetaldehyde agarose carrier, and the mixture was left at 25 ℃ with HCl to adjust the pH to 7.5.

The supernatant was sampled at mixing zero and 22h after mixing and the enzyme activity of the supernatant was measured as described previously. According to the formula, the immobilization rate of the SpyTag-LPA on the blank acetaldehyde agarose carrier is about 0% after 22 h. Meanwhile, no enzyme activity was detected by the immobilized blank acetaldehyde agarose carrier particles.

Comparative example 3: blank acetaldehyde agarose Carrier + SpyTag-LDH

Blank acetaldehyde agarose supports were prepared as in comparative example 1.

The blank acetaldehyde agarose carrier thus obtained was mixed with the SpyTag-LDH prepared in example B above in a centrifuge tube, 5mg of SpyTag-LDH was added per gram of blank acetaldehyde agarose carrier, and the mixture was left at 30 ℃ and pH 8.5.

The supernatant was sampled at mixing zero and 15h after mixing and the enzyme activity of the supernatant was measured as described previously. The immobilization rate of SpyTag-LDH on blank acetaldehyde agarose carrier was about 0% after 15h, calculated according to the formula above. Meanwhile, no enzyme activity was detected by the immobilized blank acetaldehyde agarose carrier particles.

Comparative example4: blank epoxy vector + SpyTag-GFP

A blank epoxy-based carrier was prepared as in example A by weighing 1g of epoxy-based carrier, adding deionized water and 1.25M phosphate buffer, and adjusting the pH of the system to 8-9. Shaking at 25 deg.C and 200rpm for 24h to obtain blank epoxy carrier.

The resulting blank epoxy-based carrier was mixed with SpyTag-GFP prepared as described in example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of blank epoxy-based carrier, and the mixture was placed at 28 ℃ and pH 8.5.

The supernatant was sampled at mixing zero and 20h after mixing and the fluorescence of the supernatant was measured as described previously. The immobilization rate of SpyTag-GFP on the blank epoxy-based carrier was about 0% over 20h, calculated according to the formula above. Meanwhile, the immobilized blank epoxy-based carrier particles are free of fluorescence when detected under a fluorescence microscope.

Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: the invention may be modified and equivalents substituted; any modification or partial replacement without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

SEQUENCE LISTING

<110> Beijing university of science and technology

<120> Process for producing immobilized enzyme or immobilized protein, and immobilized enzyme or immobilized protein

<130> I2019TC3529CB

<160> 27

<210> 1

<211> 372

<212> DNA

<213> Artificial Sequence

<220>

<223> gene coding sequence of oSPyCatcher

<400> 1

gggagtggtg gcagcggagg cgccatggtt gataccttat caggtttatc aagtgagcaa 60

ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattaa attctcaaaa 120

cgtgatgagg acggcaaaga gttagctggt gcaactatgg agttgcgtga ttcatctggt 180

aaaactatta gtacatggat ttcagatgga caagtgaaag atttctacct gtatccagga 240

aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 300

tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360

catatttaat ga 372

<210> 2

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> gene coding sequence of SpyTag

<400> 2

atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60

ggt 63

<210> 3

<211> 390

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene coding sequence of mSpyCatcher

<400> 3

gggagtggtg gcagcggagg cgccatggtt gataccttat caggtttatc aagtgagcaa 60

ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattcg cttctcaaaa 120

cgtgatgagg acggccgaga gttagctggt gcaactatgg agttgcgtga ttcatctggt 180

aaaactatta gtacatggat ttcagatgga caagtgcgcg atttctacct gtatccagga 240

aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 300

tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360

catatttaat gaaagggcaa aggcaaaggc 390

<210> 4

<211> 777

<212> DNA

<213> Artificial Sequence

<220>

<223> gene coding sequence of SpyTag-GFP

<400> 4

atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60

ggtatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 120

ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac 180

ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 240

cttgtcacta ctttcggtta tggtgttcaa tgctttgcga gatacccaga tcatatgaaa 300

cagcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt 360

ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 420

gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac 480

aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 540

ggaatcaaag ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca 600

gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 660

tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 720

cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaa 777

<210> 5

<211> 1149

<212> DNA

<213> Artificial Sequence

<220>

<223> gene coding sequence of SpyTag-GFP-SpyCatcher

<400> 5

atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60

ggtatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 120

ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac 180

ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 240

cttgtcacta ctttcggtta tggtgttcaa tgctttgcga gatacccaga tcatatgaaa 300

cagcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt 360

ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 420

gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac 480

aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 540

ggaatcaaag ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca 600

gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 660

tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 720

cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaaggg 780

agtggtggca gcggaggcgc catggttgat accttatcag gtttatcaag tgagcaaggt 840

cagtccggtg atatgacaat tgaagaagat agtgctaccc atattaaatt ctcaaaacgt 900

gatgaggacg gcaaagagtt agctggtgca actatggagt tgcgtgattc atctggtaaa 960

actattagta catggatttc agatggacaa gtgaaagatt tctacctgta tccaggaaaa 1020

tatacatttg tcgaaaccgc agcaccagac ggttatgagg tagcaactgc tattaccttt 1080

acagttaatg agcaaggtca ggttactgta aatggcaaag caactaaagg tgacgctcat 1140

atttaatga 1149

<210> 6

<211> 390

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene coding sequence of aPsyCatcher

<400> 6

gggagtggtg gcagcggagg cgccatggtt gataccttat caggtttatc aagtgagcaa 60

ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattaa attctcaaaa 120

cgtgatgagg acggcaaaga gttagctggt gcaactatgg agttgcgtga ttcatctggt 180

aaaactatta gtacatggat ttcagatgga caagtgaaag atttctacct gtatccagga 240

aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 300

tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360

catatttaat gaaagggcaa aggcaaaggc 390

<210> 7

<211> 372

<212> DNA

<213> Artificial Sequence

<220>

<223> gene coding sequence of cSpyCatcher

<400> 7

gggagtggtg gcagcggagg cgccatggtt gataccttat caggtttatc aagtgagcaa 60

ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattcg cttctcaaaa 120

cgtgatgagg acggccgaga gttagctggt gcaactatgg agttgcgtga ttcatctggt 180

aaaactatta gtacatggat ttcagatgga caagtgcgcg atttctacct gtatccagga 240

aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 300

tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360

catatttaat ga 372

<210> 8

<211> 1074

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene coding sequence of LPA

<400> 8

atgaatggtg aaacatcacg tccaccagct ctaggttttt catcagataa tattgctggt 60

gcttcaccag aagttgctca agctctagtt aaacattctt ctggtcaggc tggtccgtac 120

ggtactgacg aactgaccgc tcaggttaaa cgtaaattct gcgaaatctt cgaacgtgac 180

gttgaagttt tcctggttcc gaccggtact gctgctaacg ctctgtgcct gtctgctatg 240

accccgccgt ggggtaacat ctactgccac ccggcttctc acatcaacaa cgacgaatgc 300

ggtgctccgg aatttttctc taacggtgct aaactgatga ccgttgacgg tccggctgct 360

aaactggaca tcgttcgtct gcgtgaacgt acccgtgaaa aagttggtga cgttcacacc 420

acccagccgg cttgcgtttc tatcacccag gctaccgaag ttggttctat ctacaccctg 480

gacgaaatcg aagctatcgg tgacgtttgc aaatcttctt ctctgggtct gcacatggac 540

ggttctcgtt tcgctaacgc tctggtttct ctgggttgct ctccggctga aatgacctgg 600

aaagctggtg ttgacgctct gtctttcggt gctaccaaaa acggtgttct ggctgctgaa 660

gctatcgttc tgttcaacac ctctctggct accgaaatgt cttaccgtcg taaacgtgct 720

ggtcacctgt cttctaaaat gcgtttcctg tctgctcaga tcgacgctta cctgaccgac 780

gacctgtggc tgcgtaacgc tcgtaaagct aacgctgctg ctcagcgtct ggctcagggt 840

ctggaaggtc tgggtggtgt tgaagttctg ggtggtactg aagctaacat cctgttctgc 900

cgtctggact ctgctatgat cgacgctctg ctgaaagctg gtttcggttt ctaccacgac 960

cgttggggtc cgaacgttgt tcgtttcgtt acctctttcg ctaccaccgc tgaagacgtt 1020

gaccacctac taaatcaagt acgactagct gctgatcgta ctcaggaacg ataa 1074

<210> 9

<211> 1101

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene-encoding sequence of LDH

<400> 9

atgaccctgg aaatcttcga atacctggaa aaatacgact acgaacaggt tgttttctgc 60

caggacaaag aatctggtct gaaagctatc atcgctatcc acgacaccac cctgggtccg 120

gctctgggtg gtacccgtat gtggacctac gactctgaag aagctgctat cgaagacgct 180

ctgcgtctgg ctaaaggtat gacctacaaa aacgctgctg ctggtctgaa cctgggtggt 240

gctaaaaccg ttatcatcgg tgacccgcgt aaagacaaat ctgaagctat gttccgtgct 300

ctgggtcgtt acatccaggg tctgaacggt cgttacatca ccgctgaaga cgttggtacc 360

accgttgacg acatggacat catccacgaa gaaaccgact tcgttaccgg tatctctccg 420

tctttcggtt cttctggtaa cccgtctccg gttaccgcgt atggtgtata ccggggtatg 480

aaagctgctg ctaaggaggc gttcggtacc gacaatctgg aaggtaaagt tatcgctgtt 540

cagggtgttg gtaacgttgc ttaccacctg tgcaaacacc tgcacgctga aggtgctaaa 600

ctgatcgtta ccgacatcaa caaagaagct gttcagcgtg ctgttgaaga attcggtgct 660

tctgctgttg aaccgaacga aatctacggt gttgaatgcg acatctacgc tccgtgcgct 720

ctgggtgcta ccgttaacga cgaaaccatc ccgcagctga aagctaaagt tatcgctggt 780

tctgctaaca accagctgaa agaagaccgt cacggtgaca tcatccacga aatgggtatc 840

gtttacgctc cggactacgt tatcaacgct ggtggtgtta tcaacgttgc tgacgaactg 900

tacggttaca accgtgaacg tgctctgaaa cgtgttgaat ctatctacga caccatcgct 960

aaagttatcg aaatctctaa acgtgacggt atcgctacct acgttgctgc tgaccgtctg 1020

gctgaagaac gtatcgcttc tctgaaaaac tctcgttcta cctacctgcg taacggtcat 1080

gatattattt ctcgtcgtta a 1101

<210> 10

<211> 1137

<212> DNA

<213> Artificial Sequence

<220>

<223> gene coding sequence of SpyTag-LPA

<400> 10

atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60

ggtatgaatg gtgaaacatc acgtccacca gctctaggtt tttcatcaga taatattgct 120

ggtgcttcac cagaagttgc tcaagctcta gttaaacatt cttctggtca ggctggtccg 180

tacggtactg acgaactgac cgctcaggtt aaacgtaaat tctgcgaaat cttcgaacgt 240

gacgttgaag ttttcctggt tccgaccggt actgctgcta acgctctgtg cctgtctgct 300

atgaccccgc cgtggggtaa catctactgc cacccggctt ctcacatcaa caacgacgaa 360

tgcggtgctc cggaattttt ctctaacggt gctaaactga tgaccgttga cggtccggct 420

gctaaactgg acatcgttcg tctgcgtgaa cgtacccgtg aaaaagttgg tgacgttcac 480

accacccagc cggcttgcgt ttctatcacc caggctaccg aagttggttc tatctacacc 540

ctggacgaaa tcgaagctat cggtgacgtt tgcaaatctt cttctctggg tctgcacatg 600

gacggttctc gtttcgctaa cgctctggtt tctctgggtt gctctccggc tgaaatgacc 660

tggaaagctg gtgttgacgc tctgtctttc ggtgctacca aaaacggtgt tctggctgct 720

gaagctatcg ttctgttcaa cacctctctg gctaccgaaa tgtcttaccg tcgtaaacgt 780

gctggtcacc tgtcttctaa aatgcgtttc ctgtctgctc agatcgacgc ttacctgacc 840

gacgacctgt ggctgcgtaa cgctcgtaaa gctaacgctg ctgctcagcg tctggctcag 900

ggtctggaag gtctgggtgg tgttgaagtt ctgggtggta ctgaagctaa catcctgttc 960

tgccgtctgg actctgctat gatcgacgct ctgctgaaag ctggtttcgg tttctaccac 1020

gaccgttggg gtccgaacgt tgttcgtttc gttacctctt tcgctaccac cgctgaagac 1080

gttgaccacc tactaaatca agtacgacta gctgctgatc gtactcagga acgataa 1137

<210> 11

<211> 1164

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene-encoding sequence of SpyTag-LDH

<400> 11

atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60

ggtatgaccc tggaaatctt cgaatacctg gaaaaatacg actacgaaca ggttgttttc 120

tgccaggaca aagaatctgg tctgaaagct atcatcgcta tccacgacac caccctgggt 180

ccggctctgg gtggtacccg tatgtggacc tacgactctg aagaagctgc tatcgaagac 240

gctctgcgtc tggctaaagg tatgacctac aaaaacgctg ctgctggtct gaacctgggt 300

ggtgctaaaa ccgttatcat cggtgacccg cgtaaagaca aatctgaagc tatgttccgt 360

gctctgggtc gttacatcca gggtctgaac ggtcgttaca tcaccgctga agacgttggt 420

accaccgttg acgacatgga catcatccac gaagaaaccg acttcgttac cggtatctct 480

ccgtctttcg gttcttctgg taacccgtct ccggttaccg cgtatggtgt ataccggggt 540

atgaaagctg ctgctaagga ggcgttcggt accgacaatc tggaaggtaa agttatcgct 600

gttcagggtg ttggtaacgt tgcttaccac ctgtgcaaac acctgcacgc tgaaggtgct 660

aaactgatcg ttaccgacat caacaaagaa gctgttcagc gtgctgttga agaattcggt 720

gcttctgctg ttgaaccgaa cgaaatctac ggtgttgaat gcgacatcta cgctccgtgc 780

gctctgggtg ctaccgttaa cgacgaaacc atcccgcagc tgaaagctaa agttatcgct 840

ggttctgcta acaaccagct gaaagaagac cgtcacggtg acatcatcca cgaaatgggt 900

atcgtttacg ctccggacta cgttatcaac gctggtggtg ttatcaacgt tgctgacgaa 960

ctgtacggtt acaaccgtga acgtgctctg aaacgtgttg aatctatcta cgacaccatc 1020

gctaaagtta tcgaaatctc taaacgtgac ggtatcgcta cctacgttgc tgctgaccgt 1080

ctggctgaag aacgtatcgc ttctctgaaa aactctcgtt ctacctacct gcgtaacggt 1140

catgatatta tttctcgtcg ttaa 1164

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> SpyTag-GFP upstream primer

<400> 12

gaactataca aatgaaagct tgcggccgca ct 32

<210> 13

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> SpyTag-GFP downstream primer

<400> 13

ttcatttgta tagttcatcc atgccatgtg t 31

<210> 14

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Osppycatcher upstream primer

<400> 14

gcagccatgg gagtggtggc agcggagg 28

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> downstream primer of OspyCatcher

<400> 15

ccactcccat ggctgccgcg cggcacca 28

<210> 16

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> aPsycatcher upstream primer

<400> 16

aagggcaaag gcaaaggcaa gcttgcggcc gcact 35

<210> 17

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> aPsycatcher downstream primer

<400> 17

gcctttgcct ttgccctttc attaaatatg agcgtc 36

<210> 18

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> cSpycatcher (positions 28 and 37) upstream primers

<400> 18

caaaacgtga tgaggacggc cgagagttag ctggtgcaac 40

<210> 19

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> cSpycatcher (positions 28 and 37) downstream primers

<400> 19

tcctcatcac gttttgagaa gcgaatatgg gtagcactat ctt 43

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> cSpycatcher (position 64) upstream primer

<400> 20

gacaagtgcg cgatttctac ctgtatccag 30

<210> 21

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> cSpycatcher (position 64) downstream primer

<400> 21

gtagaaatcg cgcacttgtc catctgaaat 30

<210> 22

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> pET28a-SpyTag plasmid backbone upstream primer

<400> 22

aaaaggtctc agcttctaac aaagcccgaa agg 33

<210> 23

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> pET28a-SpyTag plasmid backbone downstream primer

<400> 23

ttttggtctc taccggaacc ccctgaa 27

<210> 24

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> LPA upstream primer

<400> 24

ttttggtctc tcggtatgaa tggtgaaaca 30

<210> 25

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> LPA downstream primer

<400> 25

ttttggtctc tgcttttatc gttcctgagt 30

<210> 26

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> LDH upstream primer

<400> 26

ttttggtctc tcggtatgac cctggaaatc 30

<210> 27

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> LDH downstream primer

<400> 27

ttttggtctc tgcttttaac gacgagaaat a 31

Claims

-covalently binding SpyCatcher to the carrier;

2. An immobilized enzyme or immobilized protein comprising:

-a carrier;

-SpyCatcher covalently bound to the carrier; and

-a target enzyme or target protein fused at the N-or C-terminus with SpyTag;

wherein the target enzyme or the target protein is immobilized on a carrier by covalently binding the SpyTag to the SpyCatcher.

3. A vector to which SpyCatcher is covalently bound.

4. Use of a vector to which SpyCatcher is covalently bound for selectively immobilizing a target enzyme or a target protein having SpyTag fused at the N-terminus or C-terminus from a protein mixture.

5. The method of claim 1 or the immobilized enzyme or protein of claim 2 or the vector of claim 3 or the use of claim 4, wherein the SpyCatcher has the gene coding sequence of SEQ ID No.1, or has the gene coding sequence of SEQ ID No.3, or has the gene coding sequence of SEQ ID No.6, or has the gene coding sequence of SEQ ID No. 7.

6. The method of claim 1 or 5 or the immobilized enzyme or protein of claim 2 or 5 or the use of claim 4, wherein the SpyTag has the gene coding sequence of SEQ ID No. 2.

7. The method of any one of claims 1 and 5 to 6 or the immobilized enzyme or immobilized protein of any one of claims 2 or 5 to 6 or the vector of claim 3 or 5 or the use of claim 4 or 5 or 6, wherein the vector is an epoxy-based vector or an acetaldehyde agarose vector.

8. The method according to any one of claims 1 and 5 to 7 or the immobilized enzyme or protein according to any one of claims 2 or 5 to 7 or the use according to any one of claims 4 to 6, wherein the enzyme of interest is an L-phenylserine aldolase or a leucine dehydrogenase, and the protein of interest is a green fluorescent protein, preferably the L-phenylserine aldolase having the gene coding sequence of SEQ ID No.8, and the leucine dehydrogenase having the gene coding sequence of SEQ ID No. 9.

9. An immobilized enzyme or an immobilized protein obtained by the method of any one of claims 1 and 5-8.