Artificial sensor element combination, nucleic acid encoding same, recombinant expression vector containing nucleic acid, host cell and application thereof

Abstract

The invention discloses an artificial sensor element combination, nucleic acid for coding the artificial sensor element combination, a recombinant expression vector containing the nucleic acid, a host cell and application of the artificial sensor element combination. The artificial sensor element combination comprises a polypeptide chain 1, a polypeptide chain 2 and a specific substrate, and the method is used for high-throughput detection of protein solubility, stability and enzyme activity. When the artificial sensor element combination is used for detecting protein properties, the problems of inaccuracy, false positive and the like of soluble detection of a conventional protein soluble sensor caused by dynamic characteristics of protein expression, folding and aggregation processes can be at least solved.

Description

Technical Field

[0001] The present application relates to the fields of biotechnology and biodetection technology, and in particular to an artificial sensor element combination, a nucleic acid encoding the same, a recombinant expression vector containing the nucleic acid, a host cell and applications thereof. Background Art

[0002] The soluble expression of proteins is a key step in the biosynthesis of functional proteins such as enzymes and antibodies. It is of great significance in the biotechnology and pharmaceutical industries, especially in the design and variant screening of proteins, and the production of recombinant proteins. When many exogenous proteins are expressed in host cells, they often form insoluble inclusion bodies, which affects the realization of the normal physiological functions of the proteins and is difficult to separate and purify without affecting the functions. There are many factors affecting the soluble expression of proteins, including: nucleic acid / amino acid sequence, transcription and translation speed, protein folding, host molecular chaperone network, and environmental factors such as temperature and pH. The soluble expression of heterologous insoluble proteins requires the optimization of multiple factors. In addition, the sequences generated in protein design and modification also need to be quickly screened to obtain individuals that can be soluble. Therefore, the development of a fast and effective protein solubility detection method has become one of the key issues in the field of protein engineering.

[0003] Traditional protein solubility detection methods, such as centrifugation and SDS-PAGE, Western-Blot analysis, etc., can provide relatively accurate qualitative and quantitative information, but usually require complex operations and long experimental time, and cannot perform high-throughput detection of large-scale libraries. With the development of biosensor technology, biosensor-based detection methods have shown great potential. These sensors can detect protein solubility in real time, quickly and sensitively, reduce the complexity of the experiment, and provide higher throughput. Through specific recognition elements and signal conversion elements, biosensors can convert changes in protein solubility into detectable light signals or growth rate signals, thereby achieving rapid detection of target proteins.

[0004] At present, some biosensors for protein solubility detection have been developed, of which the two main types are GFP-based fluorescent sensors and growth-coupled sensors based on growth-related proteins. A sensor based on split GFP was reported in 2005 (S. Cabantous, et al., Nat. Biotech., 23, 102-07, 2005). Due to its simple operation method and small fusion tag size, the split GFP sensor has been widely used. However, the GFP-based method has a high false positive rate due to the coupling of protein synthesis and solubility detection, so its application has poor universality. Another type of sensor coupled to growth signals is represented by split antibiotic resistance protein (K. Maxwell, et al., Protein Science, 8(9): 1908-11, 2008) or T7 RNA polymerase (T. Wang, et al., Nat. Chem. Biol., 14, 972-80, 2018). Although high-throughput detection can be performed with the help of population size differences, due to the large size of their fusion components, they are prone to affecting the intrinsic solubility of the target protein in practical applications, affecting the accuracy and universality of their detection.

[0005] Therefore, there is an urgent need to develop a protein soluble sensor and a detection method thereof that has a wide range of applications, high accuracy, and is capable of high-throughput detection. Summary of the invention

[0006] Based on this, it is necessary to provide at least one combination of artificial sensor elements, nucleic acids encoding the same, recombinant expression vectors containing the nucleic acids, host cells and applications thereof.

[0007] In a first aspect of the present application, an artificial sensor element combination is provided, which includes polypeptide chain 1 and polypeptide chain 2, and a specific substrate; and is used for high-throughput detection of protein solubility, stability and enzyme activity.

[0008] In the second aspect of the present application, a nucleic acid is provided, which comprises one or more of nucleic acid molecule 1 and nucleic acid molecule 2; the nucleic acid molecule 1 is used to encode the polypeptide chain 1 as described above, and the nucleic acid molecule 2 is used to encode the polypeptide chain 2 as described above.

[0009] In the third aspect of the present application, a recombinant vector is provided, which comprises the nucleic acid as described in the second aspect.

[0010] In a fourth aspect of the present application, a host cell is provided, comprising one or more of the nucleic acid described in the second aspect and the recombinant expression vector described in the third aspect.

[0011] In a fifth aspect of the present application, a kit is provided, comprising one or more of the nucleic acid described in the second aspect, the recombinant expression vector described in the third aspect, and the host cell described in the fourth aspect.

[0012] In a sixth aspect of the present application, a method for detecting protein solubility, stability and enzyme activity is provided, the method comprising:

[0013] Introducing a gene encoding a protein to be tested, the nucleic acid molecule 1 as described above, and the nucleic acid molecule 2 as described above into an expression system for the protein to be tested, inducing the expression of the gene and the nucleic acid molecule, and preparing a culture;

[0014] mixing the culture with a specific substrate as described above;

[0015] Analyzing the solubility or stability of the protein to be tested according to the detection signal released after mixing;

[0016] Wherein: the gene is fused with one of the nucleic acid molecule 1 and the nucleic acid molecule 2 for expression.

[0017] The artificial sensor element combination of the present application exhibits at least the following advantages when detecting protein properties (such as solubility, thermal stability, etc.):

[0018] (1) It can solve the problems of inaccuracy and false positives in soluble detection of conventional protein soluble sensors caused by the dynamic characteristics of protein expression, folding and aggregation processes.

[0019] (2) The introduction of specific substrates ensures that the sensors bind to each other and function only in the presence of specific substrates, and do not interfere with the system under other conditions. This allows precise regulation to activate solubility or stability detection only under the physiological conditions or cell states that need to be measured. Therefore, it can be widely used in various proteins and hosts.

[0020] (3) By regulating the interaction strength between complementary elements of the sensor, a series of sensors with different response dynamic ranges can be developed. The response multiples are between 1 and 15, which can be applied to a variety of application scenarios ranging from protein solubility detection, protein thermal stability detection to protein total expression detection.

[0021] (4) The protein solubility can be detected in situ within the cell. The high-throughput detection method of solubility can be developed by combining flow cytometry and high-throughput sequencing. This solves the problem that traditional soluble detection methods cannot achieve both high throughput and accuracy, and realizes high-throughput and high-precision detection of solubility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0023] Figure 1 This is a map of the soluble sensor element expression vector pEDK-ePASSmate+tag+mCherry in the E. coli system in one embodiment of the present application.

[0024] Figure 2 This is a map of the soluble transaminase detection expression vector pEDK-ePASSmate+RbTA-tag+mCherry in the E. coli system in one embodiment of the present application.

[0025] Figure 3 This is a verification of the effect of the soluble biosensor in one embodiment of the present application in four different host systems.

[0026] Figure 4 This is the SDS-PAGE result of measuring the solubility of RbTA and its mutants in the E. coli host sensor system in one embodiment of the present application.

[0027] Figure 5 This is the correlation between the sensor solubility measurement value and the solubility of multiple target proteins in one embodiment of the present application.

[0028] Figure 6 In one embodiment of the present application, the effect of the Split-GFP sensor on the solubility of RbTA and its mutants was compared.

[0029] Figure 7 This is the correlation between the protein thermal stability characterized by the soluble sensor and the SDS-PAGE and enzyme activity characterization results in one embodiment of the present application.

[0030] Figure 8 This is a comparison result of protein solubility detected by GFP sensor and PASS sensor in one embodiment of the present application.

[0031] Fig. 9 This is a diagram showing the predicted structure of a fluorescence-activated protein mutant and a representative docking structure of a fluorescent molecule and its mutation hotspots in one embodiment of the present application.

[0032] Fig.10 This is the result of high-throughput detection of the sequence library in one embodiment of the present application. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0036] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0037] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0038] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0039] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0040] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0041] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0042] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0043] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0044] In the present application, the number "1" or "2" after a term, such as "1" in "nucleic acid molecule 1", "2" in "nucleic acid molecule 2", and "1" in "recombinant expression vector 1", has no substantive meaning and is only used to distinguish the same terms.

[0045] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0046] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0047] In view of the shortcomings of the existing methods, the inventors have developed a high-performance sensor based on signal excitation (such as fluorescent protein activation) and a high-throughput detection method adapted thereto after long-term technical and practical exploration. The sensor at least includes a fusion protein consisting of a target protein to be detected and a fluorescent protein tag, a fluorescent protein complementary element, and a fluorescent activating molecule.

[0048] In the present application, an artificial sensor element combination is provided, and its function is at least to detect the properties of the protein to be tested, which includes but is not limited to protein solubility, stability (such as thermal stability) and enzyme activity. Taking the detection of protein solubility as an example, the advantage of the sensor composed of the artificial sensor element combination of the present application is at least to decouple the expression of the protein to be tested and the process of solubility detection, solve the problem of high false positive rate and poor universality of the existing method, and have the advantages of high sensitivity, high accuracy and adjustable dynamic range, and can be widely used in high-throughput solubility detection of various proteins to be tested. In addition, the sensor can realize the detection of properties such as extracellular / intracellular protein solubility and stability; in particular, its intracellular in situ detection can realize high-throughput protein solubility / stability detection by flow cytometry and high-throughput sequencing. Finally, the present application shows through experiments that the sensor is suitable for a variety of microbial hosts, proving that it has universality.

[0049] The term "protein property" in this application can be used interchangeably with "protein property", "protein property" and "protein property", including but not limited to protein expression level, and properties related to protein folding and aggregation, such as solubility, stability (such as thermal stability), and enzyme activity, etc.

[0050] For example, the sensor and the protein to be detected are co-expressed in cells, and intracellular soluble detection can be performed.

[0051] The protein to be tested in the present application is not limited, and can be, for example, a target protein commonly used in the art whose solubility, stability and other properties need to be understood, such as transaminase, luciferase, human β-amyloid protein and antibodies.

[0052] In some embodiments, the artificial sensor element combination includes polypeptide chain 1 and polypeptide chain 2, and a specific substrate; and the artificial sensor element combination is used for high-throughput detection of protein solubility, stability and enzyme activity.

[0053] The two different polypeptide chains in the present application can constitute an active protein complex that can be activated by a specific substrate to release a detection signal. Without wishing to be bound by any theory, it is believed that polypeptide chain 1 and polypeptide chain 2 constitute complementary elements of the sensor.

[0054] In some embodiments, polypeptide chain 1 and polypeptide chain 2 are from different proteins.

[0055] In some embodiments, polypeptide chain 1 and polypeptide chain 2 are separated from the same active protein. In some embodiments, polypeptide chain 1 and polypeptide chain 2 are obtained by splitting an engineered active protein.

[0056] In some embodiments, the active protein is separated into only the two polypeptide chains.

[0057] Without wishing to be bound by any theory, it is believed that after polypeptide chain 1 and polypeptide chain 2 are recombined into an active protein complex, the main function of the active protein complex is the same or similar to that of the active protein before separation. The main function, for example, at least includes the function of being activated by a specific active substrate to release a signal. In the present application, when polypeptide chain 1 and polypeptide chain 2 are separated from the same active protein, the terms "active protein" and "active protein complex" can be used interchangeably.

[0058] In some embodiments, the polypeptide chain 1 and the polypeptide chain 2 can bind to each other and be activated to emit fluorescence in the presence of the specific substrate.

[0059] The active protein in the present application may be a fluorescence activated protein. For example, it may be a fluorescence activated protein PYP, and the specific substrate is a fluorescence activated substrate. In some embodiments, the amino acid sequence of the fluorescence activated protein PYP comprises a sequence as shown in SEQ ID NO: 1. Accordingly, its specific substrate is a 4-hydroxycinnamic acid analogue having a structural formula as shown in the following formula I:

[0060] Wherein R1 and R2 are each independently selected from H, CH 3 , CH 2 CH 3 and OCH 3 The group formed.

[0061] In other embodiments, the fluorescence activated protein is, for example, a photoactive yellow protein (PYP) subjected to a point mutation to make it a photoactive yellow protein (PASS) that can be fluorescently activated by a 4-hydroxybenzylidene rhodanine analogue as shown in the following formula II:

[0062] Wherein R1 and R2 are each independently selected from H, CH 3 , CH 2 CH 3 and OCH 3 The amino acid sequence of the photoactive yellow protein mutant obtained after mutation is, for example, any one of SEQ ID NOs: 2 to 8.

[0063] The inventors creatively correlated the solubility of the protein to be tested (reflected in the accessibility of its molecules) with the fluorescent signal, thereby conveniently realizing the detection of protein solubility.

[0064] In some embodiments, the polypeptide chain 1 and the polypeptide chain 2 are polypeptides formed by specific resolution of fluorescence-activated proteins, and the specific substrate is a fluorescence-activated substrate.

[0065] In some embodiments, the specific splitting includes splitting at position 110+n of the fluorescence-activated protein to obtain a polypeptide chain 1 with a length of "110+n" amino acid residues and a polypeptide chain 2 with a length of "full length of protein Ln" amino acid residues; wherein n is an integer between 1 and 9 (including this number).

[0066] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0067] In some embodiments, the polypeptide chain 1 comprises the amino acid residues from positions 1 to (111+n) of the photoactive yellow protein or its variant (e.g., PASS) as the fluorescence-activated protein main body (PASSmate), and the polypeptide chain 2 comprises the amino acid residues from positions (112+n) to 125 of the photoactive yellow protein or its variant as the fluorescence-activated protein complementary fragment (PASStag).

[0068] In some embodiments, the photoactive protein may also be a protein having the same or similar function by subjecting the amino acid sequence shown in SEQ ID NO: 3 to mutations such as substitution, deletion, and / or addition of one or more amino acid residues.

[0069] In some embodiments, the method of mutating the photoactive protein with an amino acid sequence as shown in SEQ ID NO: 3 comprises: selecting a region of the structure that is far from the PASS-tag The sites with internal sequence entropy H(x)>0.5 were replaced with the same amino acid or consensus sequence.

[0070] In some embodiments, the mutation includes one or more of the following substitutions: A30V, M109L, K111R, L113I, S114T, G115P, G115D, G115S, D116E, S117K, S117R, V120I, and V125L. Without wishing to be bound by any theory, it is believed that the above substitutions are of the same type of amino acid substitutions and are located at the binding sites of polypeptide chain 1 and polypeptide chain 2, and can be used to adjust the detection function of the artificial sensor element for solubility, stability, and enzyme activity.

[0071] In addition, replacement, deletion and addition of individual amino acid sites on the polypeptide chain 1 and the polypeptide chain 2 that do not affect the three-dimensional structure and the binding between the two are all within the scope of protection of this patent.

[0072] In some embodiments, the amino acid sequence of the protein is shown in any one of SEQ ID NOs: 9-21.

[0073] In some embodiments, the photoactive protein may also be a protein having a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more with the amino acid sequence shown in SEQ ID NO:3 or the amino acid sequence after the above substitution, deletion, and / or addition, and having the same or similar functions. If not otherwise specified, the function at least includes being able to be fluorescently activated by a molecule represented by Formula II (R1 = H, R2 = CH3; hereinafter referred to as fluorescence-activated molecule A).

[0074] In this application, the term "identity" refers to sequence similarity to an amino acid / nucleotide sequence. The identity can be evaluated by the naked eye or by computer software such as ClustalW. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the degree of similarity between related sequences.

[0075] In the present application, a nucleic acid is also provided, which comprises one or more of nucleic acid molecule 1 and nucleic acid molecule 2; the nucleic acid molecule 1 is used to encode the above-mentioned polypeptide chain 1, and the nucleic acid molecule 2 is used to encode the above-mentioned polypeptide chain 2.

[0076] In the present application, the nucleic acid molecule 1 or the nucleic acid molecule 2 may be DNA, such as cDNA, genomic DNA or recombinant DNA, etc., which is not specifically limited here.

[0077] In some embodiments, the nucleic acid molecule 1 and the nucleic acid molecule 2 constitute a nucleic acid molecule. In some embodiments, the nucleic acid molecule encoding sequence constituted by the nucleic acid molecule 1 and the nucleic acid molecule 2 is an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 8. In some embodiments, the sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 22 to 25.

[0078] In some embodiments, the nucleic acid molecule 1 encodes the amino acid residues at positions 1 to (111+n) of the photoactive yellow protein or a mutant thereof (eg, PASS) as described above.

[0079] In some embodiments, the nucleic acid molecule 2 encodes the amino acid residues at positions (112+n) to 125 of the photoactive yellow protein or a mutant thereof as described above.

[0080] In some embodiments, the nucleic acid molecule 1 encodes the amino acid residues at positions 1 to (111+n) of the photoactive yellow protein or its variant (e.g., PASS) as described above, and the nucleic acid molecule 2 encodes the amino acid residues at positions (112+n) to 125 of the photoactive yellow protein or its variant as described above; and the nucleic acid molecule 1 and the nucleic acid molecule 2 constitute a nucleic acid molecule, and the sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 22 to 25.

[0081] In some embodiments, the nucleic acid is a combination product, comprising two independent nucleic acid molecules 1 and nucleic acid molecules 2.

[0082] In the present application, a recombinant expression vector is provided, which comprises the nucleic acid as described above.

[0083] In some embodiments, the recombinant expression vector is obtained by effectively linking the above-mentioned nucleic acid to an expression vector.

[0084] In some embodiments, the expression vector is selected from the group consisting of a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a phage and an artificial chromosome.

[0085] In some embodiments, the expression vector is a plasmid. The plasmid in the present application includes but is not limited to pETDuet-1, pcDNA3.1(+), pACYCDuet-1, pEDK, pNV18.1, pHT08, pJOE series, pJMP, pXMJ19 and pEC series plasmids.

[0086] In some embodiments, in the recombinant expression vector, nucleic acid molecule 1 is located upstream of nucleic acid molecule 2.

[0087] In some embodiments, the recombinant expression vector further comprises an insertion site for a nucleotide sequence encoding a protein to be tested.

[0088] In the present application, the term "insertion site" can be either a restriction endonuclease cleavage site or an upstream and downstream homology arm composed of a segment of DNA, etc., and is not specifically limited here.

[0089] In some embodiments, the insertion site is located downstream of the nucleic acid molecule 1.

[0090] In some embodiments, the insertion site is located between the nucleic acid molecule 1 and the nucleic acid molecule 2.

[0091] In some embodiments, the recombinant expression vector comprises nucleic acid molecule 1, insertion site and nucleic acid molecule 2 in sequence from the 5' end to the 3' end.

[0092] In some embodiments, the recombinant expression vector further comprises a nucleotide sequence encoding a linker. The nucleotide sequence encoding the linker can be optimized according to the host codon preference, which is not specifically limited here.

[0093] In the present application, the linker can be used to connect the polypeptide chain 1 with the protein to be detected, or to connect the polypeptide chain 2 with the protein to be detected.

[0094] In some embodiments, in the recombinant expression vector, the nucleotide sequence encoding the linker is located upstream of the nucleic acid molecule 2.

[0095] Without wishing to be bound by any theory, it is believed that the linker at least comprises the following functions: it can adjust the interaction strength between polypeptide chain 1 and polypeptide chain 2 in the sensor so that the sensor has different response dynamic ranges.

[0096] In some embodiments, in the recombinant expression vector, from the 5' end to the 3' end, nucleic acid molecule 1, an insertion site, a nucleotide sequence for encoding a linker and nucleic acid molecule 2 are included in sequence.

[0097] In the present application, the linker may be a conventional linker in the art for connecting different peptide segments. For example, the amino acid sequence of the linker may include, but is not limited to, 1 to 3 repeats of GGGGS, 1 to 3 repeats of GSS, 1 to 3 repeats of EAAAK, GSDGGSGGGSTS, SGGGGSGGGGS, and combinations of the above fragments.

[0098] In some embodiments, the linker is a linker with a length of 3 to 15 amino acid residues.

[0099] In some embodiments, the amino acid sequence of the linker comprises a sequence as shown in any one of SEQ ID NOs: 79-89.

[0100] In some embodiments, the recombinant expression vector further comprises a marker protein reporter gene.

[0101] The marker protein reporter genes in the present application include but are not limited to mCherry, GFP, eGFP, sfGFP, CFP and other similar functional proteins with fluorescence or other signals.

[0102] In some embodiments, the recombinant expression vector is a combination product, which comprises a recombinant expression vector 1 and a recombinant expression vector 2. The recombinant expression vector 1 comprises the nucleic acid molecule 1 as defined above, and the recombinant expression vector 2 comprises the nucleic acid molecule 2 as defined above.

[0103] In combination with the above, it can be seen that the recombinant expression vector may further include a nucleotide sequence encoding a linker. In the recombinant expression vector combination, the nucleotide sequence encoding the linker may be located in recombinant expression vector 1, for connecting the nucleotide sequence encoding the protein to be tested and nucleic acid molecule 1; or may be located in recombinant expression vector 2, for connecting the nucleotide sequence encoding the protein to be tested and nucleic acid molecule 2.

[0104] In some embodiments, the nucleotide sequence encoding the linker is located in the recombinant expression vector 2, upstream of the nucleic acid molecule 2. The recombinant expression vector 2 may also include an insertion site for the nucleotide sequence encoding the protein to be tested, the insertion site being located upstream of the nucleotide sequence encoding the linker.

[0105] In some embodiments, from the 5' end to the 3' end, the recombinant expression vector 2 comprises an insertion site, a nucleotide sequence encoding a linker, and a nucleic acid molecule 2 in sequence.

[0106] The present application also provides a host cell capable of expressing the sensor as described above.

[0107] In some embodiments, the host cell comprises one or more of the nucleic acid described above and the recombinant expression vector described above.

[0108] The host cell in the present application may be derived from bacteria, fungi or animals.

[0109] The bacteria in the present application may be selected from the group consisting of Escherichia, Rhodococcus, Bacillus, Corynebacterium, Pseudomonas, Streptomyces and Staphylococcus.

[0110] In the present application, the bacterium may be, for example, Bacillus licheniformis in the genus Bacillus.

[0111] The fungi in the present application can be selected from the group consisting of yeasts and molds.

[0112] In the present application, the yeast is, for example, Saccharomyces cerevisiae or Pichia Pastoris.

[0113] In the present application, the mold is, for example, Trichoderma reesei, Aspergillus oryzae or Aspergillus niger.

[0114] The animal in the present application may be a mammal, including but not limited to hamsters and humans.

[0115] The cells derived from mammals may be, for example, hamster ovary cells (CHO), human embryonic kidney cells (HEK293), and the like.

[0116] In some embodiments, the host cell is Escherichia coli, Rhodococcus ruber, Bacillus subtilis, or Corynebacterium glutamicum.

[0117] In the present application, a kit is also provided, which comprises one or more of the nucleic acid as described above, the recombinant expression vector as described above, and the host cell as described above.

[0118] In some embodiments, the kit further comprises a specific substrate as described above.

[0119] In some embodiments, the specific substrate is contained in a detection buffer, such as a fluorescence incubation buffer. Exemplarily, when the polypeptide chain 1 and the polypeptide chain 2 are derived from PASS, the fluorescence incubation buffer contains 100 mM PBS and 20 μM fluorescence activating molecule A, pH 7.5.

[0120] The kit in the present application can be used to detect the solubility, stability (eg, thermal stability) and other properties of the protein to be tested in an in vitro environment.

[0121] In the present application, when used for in vitro environmental detection, the sample containing the protein to be detected is not limited in form. For example, the sample can be bacterial culture medium, bacterial lysate, cell culture medium, cell lysate, etc.

[0122] The application form of the sensor described in the present application is not limited to the above-mentioned kit, and can also be in the form of a reagent, a combination of reagents, a device or equipment, etc., which is not specifically limited here.

[0123] The present application also provides the use of the above nucleic acid, recombinant expression vector or host cell in constructing a biosensor for detecting protein properties.

[0124] The applications in the present application include, but are not limited to, detection of extracellular / intracellular protein solubility, detection of extracellular / intracellular protein stability, and application of high-throughput methods in corresponding detections.

[0125] In the present application, when the biosensor is used for in vivo (eg, intracellular) protein solubility and / or stability detection, the biosensor can be directly induced to express in cells, thereby being used to detect the solubility of intracellular proteins under a specific expression state.

[0126] The present application also provides the use of one or more of the above-mentioned artificial sensor element combination, the above-mentioned nucleic acid, the above-mentioned recombinant expression vector, the above-mentioned host cell and the above-mentioned kit in high-throughput detection of protein solubility, stability and enzyme activity.

[0127] The high-throughput detection in this application may include, for example:

[0128] Fluorescence-based 24 / 48 / 96-well plate screening, flow cytometry analysis, and high-throughput sequencing.

[0129] In the present application, a method for detecting protein properties (such as protein solubility, stability and enzyme activity, etc.) is also provided, the method comprising:

[0130] The gene encoding the protein to be tested, the nucleic acid molecule 1 and the nucleic acid molecule 2 are introduced into the expression system of the protein to be tested, the expression of the gene and the nucleic acid molecule is induced, and the culture is prepared;

[0131] mixing the culture with a specific substrate;

[0132] Analyzing the solubility or stability of the protein to be tested according to the detection signal released after mixing;

[0133] Wherein: the gene is expressed in fusion with the nucleic acid molecule 1 or the nucleic acid molecule 2; the nucleic acid molecule 1, the nucleic acid molecule 2 and the specific substrate are as defined above.

[0134] The introduction of specific substrates in this application ensures that the sensors only bind to each other and work in the presence of specific substrates, and do not interfere with the system under other conditions, thereby accurately regulating the solubility or stability detection to be turned on only under the physiological conditions or cell states that need to be measured. Therefore, it can be widely used in various proteins and hosts. For example, for various target proteins such as transaminases, the solubility detection is correlated with the traditional SDS-PAGE method with an R value of >0.9. 2 Correlation coefficient, the detection range covers 0~80% solubility interval.

[0135] In the present application, a method for high-throughput detection of protein properties is also provided, the method comprising:

[0136] Constructing a gene library of the protein to be tested;

[0137] Prepare the culture medium for each protein to be tested: induce the expression of different proteins to be tested in the gene library respectively;

[0138] Preparing bacterial cells containing each protein to be tested: centrifuging the culture and removing the supernatant;

[0139] Prepare flow cytometry samples: resuspend the bacteria to an OD range allowed by flow cytometry, and add fluorescence-activated substrates respectively;

[0140] Sampling and sorting on a flow cytometer: selecting the gate where the bacteria are located, dividing the total population into n gates, setting the flow rate, and collecting the cell suspension corresponding to each gate; wherein n is an integer greater than 1;

[0141] High-throughput sequencing and analysis: revive and culture the cell suspension, extract plasmids, use primers containing Illumina dedicated sequencing adapter sequences and barcodes to amplify the fragments to be tested in the protein to be tested and perform high-throughput sequencing on the fragments to be tested; collect data, calculate the proportions f1 to fn of a specific sequence in the gates G1 to Gn, and calculate the overall signal intensity S of the specific sequence as a representation value of solubility based on the signal median A1 to An of each sorting gate; the calculation formula is:

[0142]

[0143] In the present application, a method for detecting the properties of an antibody is also provided, the method comprising:

[0144] Introducing the gene encoding the antibody, nucleic acid molecule 1 and nucleic acid molecule 2 into the antibody expression system, inducing the expression of the gene and nucleic acid molecule, and preparing a culture;

[0145] mixing the culture with a specific substrate;

[0146] Analyzing the solubility or stability of the antibody according to the detection signal released after mixing;

[0147] Wherein: the gene is expressed in fusion with the nucleic acid molecule 1 or the nucleic acid molecule 2; the nucleic acid molecule 1, the nucleic acid molecule 2 and the specific substrate are as defined above.

[0148] Some examples are provided below.

[0149] The following embodiments of the present application will be described in detail in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In the following examples, the pET series expression vectors used were purchased from Novagen, the pHT08 expression vector was purchased from MoBiTec, the pXMJ19 expression vector was purchased from Beijing Solebow Technology Co., Ltd., and the expression vectors pEDK and pNV18.1-Pa2 were from the literature (Chiba, et al., Jpn J Infect Dis 2007, 60 (1), 45-47.). Other materials, reagents, etc. used, if not otherwise specified, are obtained from commercial sources. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or conventional conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0150] Example 1 Design of fluorescence activated protein PYP mutant

[0151] Based on the analysis of the yellow fluorescent protein PYP structure (PDB: 1nwz; SEQ ID NO: 1), it was found that the chromophore binding residues of wild-type PYP and the natural chromophore 4-hydroxycinnamic acid formed a covalently linked complex with hydroxybenzylidene rhodanine substances:

[0152] The structure is similar, and a mutant C69G (SEQ ID NO: 2) with C69 mutated to Gly was constructed to accommodate the new hydroxybenzylidene rhodanine chromophore in a non-covalent form. After the mutant structure was predicted by AlphaFold2, the mutant and the chromophore were replaced by the fluorescent molecule representative (shown in the above structural formula II, where R1=H, R2=CH3) using AutoDock for molecular docking, and Gromacs was used for molecular dynamics simulation to analyze the steric effect during the molecular binding process. Then, based on the C69G mutation, the substrate binding site was found to be the target of the substrate binding site through molecular dynamics trajectory analysis and the docking conformation and binding energy analysis of the fluorescent molecule. Mutations that can eliminate the steric hindrance of the fluorescent molecule binding pocket and strengthen the protein-small molecule interaction were introduced into the range: Y94W, F96I / L, D97P, Y98T / S, Q99T / S, M100R / K, T101G / S. The above point mutations were then combined, and the six mutants with the highest binding scores to the fluorescent molecule were obtained through mutation modeling and Autodock virtual screening. The corresponding mutations are shown in the following table:

[0153] Table 1-1 List of fluorescence activated protein combination mutants

[0154]

[0155] The predicted structure of the fluorescence activated protein mutant shown in SEQ ID NO: 3 and the docking structure diagram of the representative fluorescent molecule (Formula II; R1 = H, R2 = CH3) and its mutation hot spots are shown in Fig. 9 shown.

[0156] Example 2: Construction of protein soluble biosensor

[0157] (1) Construction of fluorescent protein elements for different hosts and target protein systems

[0158] In order to realize the target protein detection function, the functional motif splitting was introduced, and the PASS protein was split at the 115th position of the fluorescence activated protein mutant shown in SEQ ID NO: 3 to obtain two parts: PASS-mate (containing the 1st to 115th positions of the PASS protein) and PASS-tag (containing the 116th to 125th positions of the PASS protein). For a specific target protein (POI), the POI and PASS-tag are connected using the Linker sequence for fusion expression when the detection element is constructed.

[0159] In order to make the fluorescent protein element more suitable for expression in Escherichia coli, Rhodococcus ruber, Bacillus subtilis, Corynebacterium glutamicum and other expression hosts, the PASS amino acid sequence (as shown in SEQ ID NO: 3) was codon optimized to obtain 4 different DNA sequences, as shown in SEQ ID NO: 22 to 25. The above gene sequences were synthesized by GenScript Biotech Co., Ltd. and connected to the pET-28a (+) expression vector to obtain expression vectors pET28a-ePASS, pET28a-rPASS, pET28a-bPASS, and pET28a-cPASS, respectively.

[0160] In addition, in order to achieve different binding strength gradients between PASS-mate and tag, Blastp was used to search for homologous sequences and construct sequence evolution alignments. The AlphaFold2 tool was used to predict the structure of PASSSPASS, and then the distance from PASS-tag in the structure was selected. The sites with internal sequence entropy H(x)>0.5 (a total of 10) were replaced with the same amino acid or consensus sequence to obtain 13 single-point mutants of PASSSPASS for subsequent activation fluorescence characterization. Specifically, the constructed single-point mutants are: A30V, M109L, K111R, L113I, S114T, G115P, G115D, G115S, D116E, S117K, S117R, V120I, V125L, numbered as PASS1~13 (SEQ ID NO:9~21), and split at position 115 in the same manner as described above to obtain the mate and tag sequences of PASS1~13. The above mutants were all constructed by PCR using bidirectional mutation primers on the PASS gene sequences optimized for 4 different hosts. The construction process is described below using the sequence for the Escherichia coli host (SEQ ID NO:22) as an example:

[0161] The above-mentioned E. coli host recombinant expression pET28a-ePASS was selected as a template, forward primers and reverse primers containing mutation sites were designed (see Table 1-2 for details), and PHANTA DNA Polymerase (Takara) was used for full plasmid PCR amplification.

[0162] The 20 μL PCR reaction system includes:

[0163] 1 μL pET28a-ePASS plasmid template (about 50 ng / μL);

[0164] 10μL 2×PHANTA DNA Polymerase;

[0165] 1.5 μL forward primer (10 μM);

[0166] 1.5 μL reverse primer (10 μM);

[0167] 6 μL ddHO 2 O;

[0168] The PCR amplification reaction cycle conditions are:

[0169] (1) Pre-denaturation: 95°C, 5 min;

[0170] (2) Denaturation: 95°C, 15 sec;

[0171] (3) Annealing: 56°C, 15 sec;

[0172] (4) Extension: 72°C, 1 kbp / min;

[0173] (5) Extension: 72°C, 8 min;

[0174] The above steps (2)-(4) were performed for a total of 34 cycles, and the final PCR product was stored at 4°C.

[0175] The recombinant plasmid obtained by PCR amplification was digested with Dpn I enzyme, transformed into E. coli DH5α competent cells, and spread on LB plate medium (peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar 15g / L, solvent is deionized water, pH=7.0) containing kanamycin (50μg / mL), and placed in a 37°C incubator for inverted culture for about 12h. Pick a single clone for sequencing verification, culture the bacterial solution after sequencing is correct, preserve the strain with 25% glycerol, and store it in a -80°C refrigerator. The recombinant expression vector containing the mutation is named pET28a-ePASS1~13.

[0176] Table 1-2. Forward primer (-F) and reaction primer (-R) sequences used to construct mutant library

[0177]

[0178]

[0179] (2) Construction of expression plasmid

[0180] The construction process of the expression plasmid includes the splitting of the fluorescence activation element PASS, the fusion of the target protein POI to be detected, and the connection of the target host expression vector. Different plasmid expression vectors are selected for different expression / detection hosts, among which: ePASS used in E. coli is expressed using the pEDK plasmid (preliminary construction of the research group), rPASS used in R. ruber is expressed using the pNV18.1 plasmid, bPASS used in B. subtilis is expressed using the pHT-08 plasmid, and cPASS used in C. glutamicum is expressed using the pXMJ19 plasmid. The following describes the expression plasmid construction method and process using the construction of ePASS in the pEDK vector as an example.

[0181] First, the plasmid backbone and the element expression reporter gene mCherry were connected. The pEDK plasmid backbone was PCR amplified using pEDK2-F and pEDK2-R, and the PCR reaction system and steps were as described above. In addition, the reporter gene mCherry fragment was amplified using pEDK2-mCherry-F and pEDK2-mCherry-R. The amplified DNA products were separated by agarose gel electrophoresis, and the DNA fragments were purified using the Gel Extraction Kit from OMEGA bio-tek. The purified pEDK backbone fragment and the mCherry gene fragment were mixed in a molar ratio of 1:4 and connected at a constant temperature of 50°C for 30 minutes using the Gibson ligation kit (CloneSmarter Technologies). Take 10 μL of the above Gibson ligation product, heat-shock transform Escherichia coli DH5α competent cells (from Transgene), spread on LB plates containing 50 μg / mL of kanamycin, and culture overnight at 37°C to grow colonies. Then pick a single colony and use universal primers DuetUp2 and T7-ter for PCR and electrophoresis verification. The colony with the correct electrophoresis band was selected for sequencing (commissioned by Azenta) to obtain the correctly sequenced Escherichia coli containing the plasmid pEDK-mCherry.

[0182] Next, connect ePASS-mate and tag to the expression vector. After selecting the appropriate POI-PASS-tag connector (using GS3 connector (GGGGS) 3 as an example), use pEDK1-F+pEDK1-R to clone the pEDK-mCherry plasmid backbone, use RBS-NcoI-F+NcoI-ePASSmate-R to clone the ePASS-mate fragment, and use pEDK-bridge-F+pEDK-bridge-R to clone the mate and tag intermediate connection fragment (including the RbTA promoter T7 promoter, etc., see Figure 2 ), use NdeI-linker-F+NdeI-ePYHPtag-R as templates to clone the linker-ePASS-tag fragment (primer sequences are shown in Table 2). After the obtained DNA fragments were separated and purified by electrophoresis according to the above method, the above-mentioned 1 skeleton and 3 fragments were mixed in a molar ratio of 1:3:3:3 and Gibson ligation was performed. After the ligation was completed, Escherichia coli DH5α competent cells were transformed, cultured, and single colonies were picked for sequencing, and finally an expression vector containing all soluble detection elements was obtained: pEDK-ePASSmate+tag+mCherry (see Figure 1 ).

[0183] Finally, the POI of the target protein to be detected is inserted, and the transaminase RbTA is selected as an example of POI. The amino acid and nucleotide sequences of the transaminase RbTA are shown in SEQ ID NO:71-72. Use Linker-RbTA-F+pEDK-RbTA-R to clone the pEDK-ePASSmate+tag+mCherry plasmid backbone constructed above, and use RbTA-F+RbTA-R to clone the POI fragment (RbTA) without a stop codon. After electrophoretic separation and purification, the above-mentioned backbone and fragment are mixed in a molar ratio of 1:4 and Gibson ligation is performed. After the connection is completed, the Escherichia coli DH5α competent cells are transformed, cultured, and single colonies are picked for sequencing, and finally a biosensor expression system that can detect the intracellular expression of soluble POI is obtained: pEDK-ePASSmate+RbTA-tag+mCherry (see Figure 2 ).

[0184] In addition, for the four PASSes and their different mutants used in different hosts, primers were designed according to the above process to construct corresponding expression systems on different expression vectors.

[0185] Table 2. Primers used in the construction of expression plasmids

[0186]

[0187] Example 3: Validation of protein soluble biosensors in different host systems

[0188] (1) Preparation of cells expressing sensor elements

[0189] The use of protein soluble biosensors first requires obtaining host cells that express sensor elements. For E. coli hosts, E. coli cells containing the soluble biosensor expression vector pEDK-ePASSmate+RbTA-tag+mCherry containing specific mutants were inoculated into 5mL LB liquid culture medium (peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solvent is deionized water, pH=7.0) containing kanamycin (50μg / mL) and cultured overnight at 37°C. The harvested bacterial liquid was extracted with the Plasmid mini kit I plasmid extraction kit from OMEGA bio-tek to obtain the expression plasmid in the corresponding host.

[0190] The expression plasmid was transformed into competent cells E.coli BL21 (DE3) using the heat shock method, coated with LB plate medium containing kanamycin, and cultured overnight at 37°C, and then a single colony was picked for gene sequencing (commissioned by Azenta). The single colony with correct sequencing was inoculated into LB liquid medium containing kanamycin, and cultured at 37°C and 200rpm for 12h to obtain a seed solution expressing transaminase or its mutant. The seed solution was inoculated into a shake flask containing 1000mL LB liquid medium (containing 50μg / mL kanamycin) at a ratio of 1% (v / v, volume percentage), cultured at 37°C and 200rpm for 3h, IPTG was added at a final concentration of 0.5mM, and cultured at 30°C and 200rpm for 12h to induce protein expression to obtain a fermentation broth. The fermentation broth was centrifuged at 8000×g for 20min and the cells were collected to obtain cells expressing soluble biosensor elements and protein transaminase to be tested.

[0191] For the R. ruber host, the expression plasmid pNV18.1-Pa2-rPASSmate+RbTA-tag+mCherry was extracted from E. coli using the same steps as above. The plasmid was transferred into R. ruber TH by electroporation (Jiao, et al., New Biotechnology, 44:41-49, 2018), and the transformants were cultured on a plate medium containing 6 mg / L tetracycline (glucose 10 g / L, yeast extract 3 g / L, NaCl 1 g / L, K 2 HPO 4 ·3H 2 O 2g / L, MgSO 4 7H 2 O0.2g / L, agar 15g / L) for screening. After culturing at 28°C for 24h, single colonies were picked for gene sequencing. The single colonies with correct sequencing were inoculated into 6mg / L Rhodococcus seed culture medium (glucose 20g / L, yeast extract 1g / L, peptone 1g / L, K 2 HPO 4 ·3H 2 O 0.5g / L, KH 2 PO 4 0.5g / L, MgSO 4 7H 2 O 0.5 g / L, pH 7.5), cultured at 37 ° C, 200 rpm for 24 h, and then transferred to the Rhodococcus fermentation medium (glucose 20 g / L, yeast extract 5 g / L, urea 6 g / L, K 2 HPO 4 ·3H 2 O 2.28g / L, KH2 PO 4 0.866 g / L, MgSO 4 7H 2 O 1g / L, sodium glutamate 0.5g / L, Co 2+ 50ppm) and cultured at 28°C, 200rpm for 48h. The bacterial solution was centrifuged at 8000×g for 20min before the cells were harvested.

[0192] For the Bacillus subtilis (B. subtilis) host, the expression plasmid pHT08-bPASSmate+RbTA-tag+mCherry was extracted from Escherichia coli using the same steps as above. The extracted plasmid with the sensor element was transformed into the competent cells of Bacillus subtilis THY-7 by electroporation. Among them, the preparation and electroporation of Bacillus subtilis THY-7 competent cells adopt the method in Chinese patent document CN105400784A. The transformants were screened by culturing overnight at 37°C using LB plate culture medium containing 50mg / L ampicillin. The single colonies grown were sequenced, and the single colonies with correct sequencing were inoculated in LB liquid culture medium and cultured for 16h at 37°C and 200rpm to obtain seed liquid. The seed solution was transferred to 20 mL of fermentation medium containing 50 mg / L ampicillin (sugar 30-100 g / L, inorganic nitrogen source 10-50 g / L, organic nitrogen source 0.5-3 g / L, KH 2 PO 4 0.1-1g / L, Na 2 HPO 4 12H 2 O 0.5-0.3 g / L, CaCl 2 0.002-0.01g / L, MnSO 4 ·H 2 O 0.002-0.01g / L, FeSO 4 7H 2 O 0.002-0.01g / L, pH 6.5-7.5), cultured at 37°C, 200rpm for 2-6h, added 1mM IPTG for induction, and cultured for 24h to obtain the expression bacterial solution. The bacterial solution was centrifuged and the cells were harvested as described above.

[0193] For the Corynebacterium glutamicum (C. glutamicum) host, the expression plasmid pXMJ19-cPASSmate+RbTA-tag+mCherry was extracted from Escherichia coli using the same steps as above. The extracted plasmid was transferred into Cg0 competent cells by electrotransformation (specific competent preparation and transformation methods are shown in Chinese patent document CN202111338352A). The transformants were screened using LB plate culture medium containing 5 mg / L chloramphenicol to obtain single colonies with correct sequencing. The recombinant bacteria were inoculated in LB liquid culture medium, cultured at 30°C and 200rpm for 12-16h, and then accessed to the fermentation medium at a volume ratio of 5%, and cultured at 30°C and 200rpm for 3h, and IPTG was added at a final concentration of 1mM for induction, and the culture was continued for 48h. The bacterial liquid was centrifuged and the cells were harvested as described above.

[0194] (2) Characterization of soluble biosensors

[0195] The cells harvested in the above process were resuspended and washed twice with PBS buffer (pH = 7.5) (Beijing Solebow Technology Co., Ltd.) and finally diluted to OD 600 =1.0~3.0 to obtain the bacterial suspension to be tested. Add the fluorescent activating molecule A with a final concentration of 10~100 μM and 1% v / v DMSO to 2 mL of the bacterial solution to be tested, shake at 1000 rpm for 30 min at 30°C, and then let stand on ice for 10 min. Transfer 200 μL of the reaction solution to a black 96 ELISA plate, and use an Infinite 200Pro ELISA reader (manufacturer TECAN, Switzerland) to measure the fluorescence (Ex / Em=480 / 540 nm).

[0196] For four different application hosts (E. coli BL21 (DE3), R. ruber TH, B. subtilis THY-7, C. glutamicum Cg0), the transaminase RbTA wild type WT (basically soluble) and mutant F228K (insoluble) were used as inserted POI to verify the construction of the sensor. The corresponding PASSmate and tag used the elements and plasmid vectors corresponding to the specific host. The solubility of the fluorescence characterization according to the above process is as follows Figure 3 As shown, different POI insertions all have fluorescence compared to the negative control NC, indicating that each component is normally expressed in the host bacteria. The significantly higher fluorescence of WT compared to the mutant F228K indicates the sensor's ability to characterize the intracellular solubility of POI.

[0197] Example 4: Using biosensors to detect the intracellular solubility of multiple target protein POIs

[0198] Transaminase RbTA and its 23 mutants with different solubility, firefly luciferase Fluc and its 12 mutants with different solubility, human β-amyloid protein Aβ42 / 40 and its 16 mutants with different aggregation degrees were selected as POI (specific mutants are shown in Table 3), and a soluble biosensor based on the wild type of ePASS was constructed in the E. coli system according to the method in Example 2, and the flexible GS3 linker (amino acid sequence: (GGGGS)) was selected as the linker sequence. 3 ). After culturing and harvesting the cells using the method in Example 3, on the one hand, the fluorescence of the fluorescence-activated molecule A was measured according to the above process, and 200 μL of the same sample bacterial solution was taken using a microplate reader to measure the mCherry fluorescence (Ex / Em=535 / 620 nm) as the internal standard of POI protein expression, and the expression amount was normalized using the fluorescence of the fluorescence-activated molecule A / mCherry fluorescence as the protein solubility measurement value obtained by the sensor; on the other hand, part of the bacteria was resuspended in PBS buffer to OD 600 =3.0~4.0, add 1U / mL lysozyme (Yisheng Biotechnology Co., Ltd.) for treatment, and then use an ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) to disrupt at 4°C, with a power of 30%, ultrasound on for 2s and off for 3s, and a total disruption time of 20min. Centrifuge the cell disruption solution at 13000×g for 20min at 4°C to obtain the supernatant and precipitate. Separate and wash the precipitate and resuspend it in an equal volume of PBS buffer to obtain a precipitate resuspension. Take the supernatant and precipitate resuspension for SDS-PAGE to characterize the soluble and insoluble expression of the target protein (such as Figure 4 ). After using a gel imager to photograph the electrophoresis gel, ImageJ was used to process the image and obtain the percentage of the specific POI soluble component (S) and insoluble component (P) in the total POI expression as the true value of protein solubility. Figure 4 The positions of the bands of the test protein RbTA and its mutants, the fusion proteins of ePASS-tag (~40 kDa), and ePASS-mate (~10 kDa) are marked.

[0199] Table 3:

[0200]

[0201]

[0202] The normalized fluorescence data obtained from the above determination process were linearly regressed with the POI solubility data read from the SDS-PAGE bands to obtain the fitting R 2 For transaminase RbTA, firefly luciferase Fluc and human β-amyloid protein Aβ42 / 40, the values ​​were: >0.90, >0.90, >0.80 ( Figure 5 ). It can achieve accurate quantification of the solubility of intracellular target proteins.

[0203] In order to illustrate the advantages of the present invention, the present invention uses a split green fluorescent protein sensor (Split-GFP) method for comparison, and the test case uses transaminase and its mutants. The construction method is carried out with reference to the literature Cabantous, S. et al., Nat Biotechnol 23, 102–107 (2005). Specifically, a 15-amino acid GFP fragment (GFP11) is fused with the test protein RbTA and its mutants, and the detection element of GFP1-10 is expressed separately to form expression plasmids pTET-GFP11 and pET-GFP1-10, and expression tests are carried out in the host E. coli BL21 (DE3), and the correlation between GFP fluorescence and protein solubility is evaluated. The results are as follows Figure 6 As shown, for the protein transaminase to be tested, this method has serious false positives and cannot correlate the fluorescent signal with the solubility level of the protein.

[0204] Example 5: Thermal stability detection of target protein POI using biosensors

[0205] Soluble sensors can be used to characterize the solubility / aggregation of proteins under heat shock conditions to detect the thermal stability of proteins. The above-mentioned Escherichia coli soluble sensor system was selected, and firefly luciferase Fluc with different thermal stabilities was inserted as the target protein to be detected. According to the construction method in Example 2, the corresponding soluble detection system was obtained: pEDK-ePASSmate+Fluc-tag+mCherry, where Fluc represents luciferase and its different mutants (a total of 11). Among them, the amino acid and nucleotide sequences of Fluc are shown in SEQ ID NO:73 and 74, and the mutants involved are shown in the aforementioned Table 3. The constructed system was used to prepare protein expression cells according to the method described in Example 3 to obtain OD 600 =1.0~4.0 cell suspension was placed at 4℃ for later use.

[0206] For each Fluc variant, 5 mL of the cell suspension was heat-shocked at 37°C for 10 min to induce thermal deformation and aggregation of the protein (heat-shock group), and then allowed to stand at 4°C for 10 min to equilibrate the temperature; another 5 mL of each group was kept at 4°C as a control (control group). For the heat-shock group and control group samples of each mutant, the normalized fluorescence data [fluorescence-activated molecule A / mCherry] were determined according to the method in Example 4, and the protein solubility readings were determined by SDS-PAGE. In addition, to further ensure the accuracy of the method, the Single-Luciferase (Firefly) Reporter Assay Kit (TransGen Biotech Co., Ltd) was used to detect the luciferase activity of cells in the heat shock group and the control group, which was used as an enzyme activity indicator for thermal stability. The specific measurement steps are described in Chinese patent CN117603900 A. For the above three characterizations, the ratio of the measured value of the heat shock group to the control group was calculated as a measurement of protein thermal stability by a specific method, specifically:

[0207] Soluble Sensors:

[0208] SDS-PAGE protein electrophoresis:

[0209] Luciferase activity:

[0210] The protein thermal stability characterization effect of the sensor is as follows Figure 7 shown. Figure 7 The left half shows the correlation between the thermal stability of the sensor and the SDS-PAGE electrophoresis results. Figure 7 The right half shows the correlation between the thermal stability of the sensor and the luciferase activity assay results. The comparison results of the protein solubility detected by the GFP sensor and the PASS sensor are shown in Figure 8 It can be found that the protein thermal stability results detected by the soluble sensor can be accurately correlated with the traditional methods based on SDS-PAGE or enzyme activity (R 2 ~0.9).

[0211] Example 6: Effect verification of sensors constructed with different connectors

[0212] For different application scenarios, different POI-tag connecting joints need to be selected to adjust the affinity between the sensor PASSmate and the tag to adapt to the detection requirements of different target proteins or host systems. Taking the soluble sensor system in the Escherichia coli host as an example, the effect of different joints on the sensor performance was verified. In the pEDK-ePASSmate+RbTA-tag+mCherry detection system, the POI-tag joint was replaced with one of the following joints, and the RbTA wild type and mutant F228K were used to characterize the solubility distinction effect. For the construction of RbTA wild type and mutant F228K under each joint system, the process is shown in Example 2. The ratio of the normalized fluorescence intensity [fluorescence activated molecule A / mCherry] of the RbTA wild type and the F228K mutant under a specific joint is used to characterize the solubility distinction effect of the sensor, and the calculation formula is as follows:

[0213] Solubility differentiation effect

[0214] The measurement results are shown in Table 4.

[0215] Table 4. Differentiation effect of sensor solubility characterization under different linker systems

[0216]

[0217] In Table 4, each linker system shown is a sensor system obtained by replacing the linker amino acid sequences of RbTA and ePASStag directly in the pEDK-ePASSmate+RbTA-tag+mCherry system as shown in the table. In the solubility discrimination effect column, one plus sign "+" indicates that the WT / F228K ratio is 2 to 3 times; two plus signs "++" indicate that the ratio is 3 to 5 times; three plus signs "+++" indicate that the ratio is 5 to 10 times.

[0218] The above results show that different linkers can be used in the sensor to distinguish between soluble proteins (wild type) and insoluble proteins (F228K), but due to the different affinity of the complementary elements, there are different distinction effects. By adjusting the linker sequence, the affinity of the sensor can be fine-tuned, so that it can be applied to different protein solubility detection scenarios.

[0219] Example 7: Effect verification of sensors constructed with different fluorescent protein element mutants

[0220] Similarly, the use of different fluorescent protein element mutants can achieve the effect of regulating the affinity of complementary elements to a greater extent, and construct protein soluble sensors with different usage characteristics. The following still takes the soluble sensor system in the Escherichia coli host as an example to verify the effect of different ePASS mutants on sensor performance. In the pEDK-ePASSmate+RbTA-tag+mCherry detection system, ePASS is replaced with one of the 13 mutants with different binding strength gradients designed in Example 2 (ePASS1~13), and the RbTA wild type and mutant F228K are used to characterize the solubility distinction effect. For the construction of RbTA wild type and mutant F228K under each PASS element system, the process is as shown in Example 2, and the linker sequence uses (G4S)3. Also as in Example 6, the ratio of the normalized fluorescence intensity [fluorescence activated molecule A / mCherry] of the RbTA wild type and the F228K mutant under a specific PASS mutant is used to characterize the solubility distinction effect of the sensor, and the calculation formula is the same as described above.

[0221] In order to show the difference with wild-type ePASS, the solubility differentiation effect d of each mutant was compared with the wild-type ePASS result d ePASSAfter the difference is made, normalization is performed to obtain the relative solubility differentiation effect change Δd / d ePASS , the calculation formula is as follows:

[0222] Relative solubility differentiates effect changes:

[0223] The results are shown in Table 5.

[0224] Table 5. Changes in the discrimination effect of sensor solubility characterization under different PASS mutant elements

[0225]

[0226]

[0227] In Table 5, each mutant shown is a mutant obtained after a specific mutation occurs in the corresponding amino acid position in the amino acid sequence of the wild-type transaminase shown in SEQ ID NO: 3. In the relative solubility discrimination effect change column Δd%, one plus sign "+" indicates an increase of 0-50% compared with the wild-type ePASS; two plus signs "++" indicate an increase of 50-100%; three plus signs "+++" indicate an increase of more than 100%; one minus sign "-" indicates a decrease of 0-25% compared with the wild-type ePASS, two minus signs "--" indicate a decrease of 25-50%, and three minus signs "---" indicate a decrease of more than 50%.

[0228] The above results show that different PASS mutant elements can be used in the sensor to distinguish between soluble proteins (wild type) and insoluble proteins (F228K), but due to the different affinities of the complementary elements, there are different distinction effects. By adjusting the PASS element sequence, the affinity of the sensor can be fine-tuned, so that it can be applied to different protein solubility detection scenarios.

[0229] Example 8: Effect verification of sensors constructed by different fluorescent protein splitting methods

[0230] In addition, the use of fluorescent proteins (such as PASS) to form mate and tag elements split at different positions to construct a sensor system will also produce different detection effects. The following still takes the soluble sensor system in the Escherichia coli host as an example to verify the effect of different ePASS splitting on sensor performance. According to the splitting site amino acid numbering 111 to 119, the corresponding ePASS-mate and tag sequences are obtained, and the corresponding pEDK-ePASSmate+RbTA-tag+mCherry detection system is constructed according to the above method, and the linker sequence is selected (G4S) 3. Consistent with Example 6, the RbTA wild type and mutant F228K are used to characterize the soluble differentiation effect. The measurement results are shown in Table 6.

[0231] Table 6. Differentiation effect of solubility characterization of sensors constructed by different fluorescent protein splitting methods

[0232]

[0233] In the solubility differentiation effect column of Table 6, one plus sign "+" indicates that the WT / F228K ratio is 2 to 3 times; two plus signs "++" indicate that the ratio is 3 to 5 times; three plus signs "+++" indicate that the ratio is 5 to 10 times; - indicates that the system has no fluorescence or the ratio is close to 1.

[0234] Example 9: High-throughput detection of protein solubility using biosensors

[0235] Since the protein soluble sensor in this application can characterize the soluble expression of proteins in cells, a high-throughput detection method for protein solubility based on this soluble biosensor was developed by combining flow cytometry and high-throughput sequencing technology. The method is mainly divided into three steps:

[0236] (1) Construction of gene library; (2) Library cultivation and screening; (3) High-throughput sequencing and analysis. The following uses the soluble detection system pEDK-ePASSmate+RbTA-tag+mCherry for transaminase RbTA in Escherichia coli as an example to illustrate the specific implementation process.

[0237] (1) Construction of gene library

[0238] First, obtain a gene library containing all target protein variants to be tested. Entrust Jinweizhi Biotechnology Co., Ltd. to synthesize the N-segment random mutation library of transaminase RbTA and connect it to the pET-28a(+) plasmid. Use the plasmid of the above mutation library as a template, and use pEDK-RbTAup-F+pEDK-RbTA-R primers to perform PCR amplification of the target gene fragment (see Example 2 for steps), and obtain a DNA solution of the target gene library after electrophoresis separation and purification. In addition, the plasmid backbone pEDK-ePASSmate+tag+mCherry( Figure 1 ) was used as a template, and pEDK-HTS-F+pEDK-HTS-R was used for PCR amplification of the backbone. After electrophoresis separation and purification, a linearized plasmid backbone was obtained. Gibson ligation was performed and E. coli BL21 (DE3) competent cells were transformed using the method described in Example 2, and LB plate medium containing kanamycin was used to screen the correctly connected colonies to obtain a transformation library plate.

[0239] Add 2 mL of LB liquid medium containing 50 mg / L kanamycin to each transformation library plate, use a spreader to evenly scrape the plate colonies and mix to obtain a bacterial suspension, and collect the bacterial suspension. Take 500 μL of the bacterial suspension and add an equal volume of 50% glycerol for library preservation, and the remaining solution is used for subsequent library culture.

[0240] (2) Library cultivation and screening

[0241] The bacterial solution obtained above was inoculated into 20mL LBK liquid culture medium with an initial OD600 of 0.02, and biological parallel was set. In order to stabilize the physiological state of bacteria before flow sorting, the library bacterial solution needs to be inoculated three times continuously. Specifically, after the first inoculation at 37°C, 200rpm for 16h, it was inoculated again at OD600 = 0.02 into 20mL LB liquid culture medium containing 50mg / L kanamycin. After the third inoculation, the bacterial solution was cultured at 37°C, 200rpm for about 3h (OD600 = 0.6 ~ 0.8), a final concentration of 0.5mM IPTG was added and transferred to 16°C for induction for 16-20h.

[0242] After induction, harvest the bacterial solution. Take 500 μL of bacterial solution into a sterile EP tube and centrifuge at 8000×g at 4°C to remove the culture medium. Resuspend the cells in 500 μL of ice-cold sterile PBS buffer under sterile conditions, and then dilute the cells with PBS buffer to the OD range allowed by flow cytometry (OD 600 =0.1~0.2), and use a cell sieve to filter out large cell clusters. Before the flow sorting experiment, add the fluorescent activation molecule A at a final concentration of 20μM to the diluted bacterial solution, mix well and let it stand at room temperature for more than 1h to obtain the flow sample, and then store it in a 4℃ refrigerator / on ice.

[0243] The prepared samples were loaded and sorted using a FACS AriaSORP (BD Biosciences) flow cytometer. First, the FSC-Area and SSC-Area channels were used to select the gate where the target host E. coli was located. Then, the parallel samples to be tested were loaded separately, and the voltages of the FITC-Area (corresponding to the green fluorescence excited by the sensor) and PE-Texas Red-Area (corresponding to the red fluorescence of mCherry) channels were adjusted to keep the signals within a reasonable range. The negative and positive areas of the two channels were divided using a cross gate to accurately identify the double-positive communities in the sample. Furthermore, for the double-positive communities, the total population was evenly divided into 12 gates according to the fluorescence signal ratio of the two channels of the FITC-Area / PE-Texas Red-Area signal (fluorescence activated molecule A / mCherry). The collection flow rate was adjusted to 1.0, and the samples corresponding to the 12 gates were collected, named P1 to P12, until each gate collected >250,000 cells and stopped.

[0244] (3) High-throughput sequencing and analysis

[0245] The cell suspensions of the 12 gates collected were added to an equal volume of 2×LB medium for recovery, and cultured overnight at 37°C and 200rpm for about 12 hours. The plasmids of the bacterial liquids of G1 to G12 were extracted respectively. The obtained plasmid was used as a template, and the P7-end and P5-end primers (RbTA-P7-F and RbTA-P5-R, respectively) containing the Illumina dedicated sequencing adapter sequence were used to PCR amplify the fragment to be tested in the RbTA sequence, and the barcodes of G1 to G12 were added to the primers (barcode: AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG). After the PCR amplified fragments were separated and recovered by electrophoresis as described in Example 2, Anshengda Biotechnology Co., Ltd. was commissioned to use qubit for sample quality inspection, and then high-throughput sequencing was performed on the machine using Illumina nova6000. The collected data were screened to calculate the proportion f of a specific sequence i in the G1 to G12 gates. 1 ~f 12 , according to the signal median A of each sorting gate 1 ~A 12 , the overall signal intensity Si of sequence i is calculated cumulatively as the representation value of solubility. The specific calculation formula is as follows:

[0246] For a particular sequence i:

[0247] Table 7. Primers used in high-throughput detection

[0248]

[0249] The results of high-throughput detection of sequence libraries are as follows Fig.10 The results showed that the combinatorial library composed of different soluble mutants showed a cell individual sensor fluorescence distribution related to protein solubility in flow cytometry, and could be accurately distinguished by flow cytometry and used for downstream high-throughput sequencing analysis.

[0250] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0251] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. An artificial sensor element combination, characterized in that: It includes polypeptide chain 1 and polypeptide chain 2, and a specific substrate; and is used for high-throughput detection of protein solubility, stability and enzyme activity; Optionally, the polypeptide chain 1 and the polypeptide chain 2 can bind to each other and be activated to emit fluorescence in the presence of the specific substrate.

2. The artificial sensor element combination according to claim 1, characterized in that: The polypeptide chain 1 and the polypeptide chain 2 are polypeptides formed by specific splitting of the fluorescence-activated protein, and the specific substrate is a fluorescence-activated substrate; Optionally, the fluorescence activated protein is photoactive yellow protein PYP or a mutant thereof; The amino acid sequence of the photoactive yellow protein PYP or its mutant is shown in any one of SEQ ID NOs: 1 to 8.

3. The artificial sensor element combination according to claim 2, characterized in that: The specific splitting includes splitting at position 110+n of the fluorescence activated protein to obtain a polypeptide chain 1 having a length of "110+n" amino acid residues and a polypeptide chain 2 having a length of "the full length of the protein Ln" amino acid residues; wherein n is an integer between 1 and 9; Optionally, the same type of amino acid replacement is performed at the binding sites of the polypeptide chain 1 and the polypeptide chain 2 to adjust the detection function of the artificial sensor element for solubility, stability and enzyme activity; based on the amino acid sequence shown in SEQ ID NO:3, the same type of amino acid replacement is selected from the group consisting of A30V, M109L, K111R, L113I, S114T, G115P / S / D, D116E, S117K / R, V120I / L and V125L.

4. The artificial sensor element combination according to claim 2 or 3, characterized in that: The fluorescence activated substrate is selected from the group consisting of 4-hydroxycinnamic acid analogs and 4-hydroxybenzylidene rhodanine analogs having the structures shown below respectively; wherein R1 and R2 are each independently selected from the group consisting of H, CH3, CH2CH3 and OCH3.

5. A nucleic acid, characterized in that The nucleic acid comprises one or more of nucleic acid molecule 1 and nucleic acid molecule 2; the nucleic acid molecule 1 is used to encode polypeptide chain 1, the nucleic acid molecule 2 is used to encode polypeptide chain 2, and the polypeptide chain 1 and the polypeptide chain 2 are defined as any one of claims 1 to 4; Optionally, the nucleic acid is a combination product, comprising the nucleic acid molecule 1 and the nucleic acid molecule 2.

6. A recombinant expression vector comprising the nucleic acid according to claim 5; Optionally, the recombinant expression vector further comprises a nucleotide sequence encoding a linker; the linker is used to connect the polypeptide chain 1 with the protein to be tested, or to connect the polypeptide chain 2 with the protein to be tested.

7. The recombinant expression vector according to claim 6, characterized in that The joint satisfies one or more of the following conditions (1) to (4): (1) The linker comprises 3 to 15 amino acid residues; (2) the nucleotide sequence encoding the linker is located at the 5' end of the nucleic acid molecule 2; (3) the amino acid sequence of the linker is shown in any one of SEQ ID NOs: 79 to 89; and, (4) Used to connect the polypeptide chain 2 with the protein to be tested.

8. The recombinant expression vector according to claim 7, characterized in that The recombinant expression vector also comprises an insertion site for a nucleotide sequence encoding a protein to be tested; Optionally, the insertion site is located at the 3' end of the nucleic acid molecule 1.

9. The recombinant expression vector according to any one of claims 6 to 8, characterized in that The recombinant expression vector is a combination product, comprising a recombinant expression vector 1 and a recombinant expression vector 2, wherein the recombinant expression vector 1 and the recombinant expression vector 2 respectively comprise the nucleic acid molecule 1 and the nucleic acid molecule 2 as defined in claim 5.

10. A host cell, characterized in that It comprises the nucleic acid according to claim 5 and one or more of the recombinant expression vectors according to any one of claims 6 to 9; Optionally, the host cell is derived from bacteria, fungi or animal cells; Wherein, the bacteria may be derived from Escherichia, Rhodococcus, Bacillus, Corynebacterium, Pseudomonas, Streptomyces, Staphylococcus; The fungus may optionally be derived from a yeast or a mold; The animal cells may be derived from hamster ovary cells (CHO) or human embryonic kidney cells (HEK293).

11. A kit, characterized in that It comprises one or more of the nucleic acid according to claim 5, the recombinant expression vector according to any one of claims 6 to 9, and the host cell according to claim 10; Optionally, the kit further comprises a specific substrate, wherein the specific substrate is as defined in any one of claims 1 to 4.

12. Use of one or more of the artificial sensor element combination according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant expression vector according to any one of claims 6 to 9, the host cell according to claim 10, and the kit according to claim 11 in high-throughput detection of protein solubility, stability and enzyme activity; Optionally, high throughput detection includes: Fluorescence-based 24 / 48 / 96-well plate screening, flow cytometry analysis, and high-throughput sequencing.

13. A method for detecting protein solubility, stability and enzyme activity, characterized in that: The method comprises: The gene encoding the protein to be tested, the nucleic acid molecule 1 and the nucleic acid molecule 2 are introduced into the expression system of the protein to be tested, the expression of the gene and the nucleic acid molecule is induced, and the culture is prepared; mixing the culture with a specific substrate; Analyzing the solubility or stability of the protein to be tested according to the detection signal released after mixing; Wherein: the gene is expressed in fusion with one of the nucleic acid molecule 1 and the nucleic acid molecule 2; the nucleic acid molecule 1 and the nucleic acid molecule 2 are as defined in claim 5, and the specific substrate is as defined in any one of claims 1 to 4.

14. The method according to claim 13, characterized in that The method comprises: Constructing a gene library of the protein to be tested; Prepare the culture medium for each protein to be tested: induce the expression of different proteins to be tested in the gene library respectively; Preparing bacterial cells containing each protein to be tested: centrifuging the culture and removing the supernatant; Prepare flow cytometry samples: resuspend the bacteria to an OD range allowed by flow cytometry, and add fluorescence-activated substrates respectively; Sampling and sorting on a flow cytometer: selecting the gate where the bacteria are located, dividing the total population into n gates, setting the flow rate, and collecting the cell suspension corresponding to each gate; wherein n is an integer greater than 1; High-throughput sequencing and analysis: revive and culture the cell suspension, extract plasmids, use primers containing Illumina dedicated sequencing adapter sequences and barcodes to amplify the fragments to be tested in the protein to be tested and perform high-throughput sequencing on the fragments to be tested; collect data, calculate the proportions f1 to fn of a specific sequence in the gates G1 to Gn, and calculate the overall signal intensity S of the specific sequence as a representation value of solubility based on the signal median A1 to An of each sorting gate; the calculation formula is:

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