CN114591442B - Light-regulated protease tool and matched substrate thereof - Google Patents
Light-regulated protease tool and matched substrate thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention belongs to the technical field of molecular biology, and particularly provides a protease tool regulated and controlled by light and a matched substrate thereof. The protease means comprises an HIV-1 protease mutant and a Vivid protein domain; the Vivid protein domain is linked to the HIV-1 protease mutant by a flexible peptide; the HIV-1 protease mutant is obtained by mutating an amino acid site which mediates dimerization of HIV-1 protease under natural conditions. The invention changes the protease tool from a heterodimer form to a homodimer form, reduces two protease tool elements which are required to be transferred into the protease tool during cell experiments into one protease tool element, shortens the length of the amino acid sequence of the protease tool, and has more compact structure. In addition, when the protease tool provided by the invention is used together with a matched substrate, the control of the signal path of the artificial signal input in the mammalian cells can be realized.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a light-regulated protease tool and a matched substrate thereof.
Background
The artificial intervention is carried out on the intracellular signal path through the synthetic biological means, so that the artificial control of the cell behavior is realized, and the method has important clinical diagnosis and treatment values. Proteases have the function of altering the state of a protein substrate by hydrolyzing peptide bonds, including losing the original activity of the hydrolyzed protein substrate and removing the original inhibitory domains in the substrate to allow downstream activity of the protein substrate. Proteases can therefore be used as an effective molecular tool in the intervention of intracellular signal transduction. In order to realize the artificial control of the hydrolytic activity of the protease, engineering modification of the protease is a common technical means. The current common strategy is to split the complete protease into two domains, and then flexibly link the two protease domains to the inducible heterodimerization domains, respectively. Six split-type inducible proteases have been published that have been engineered from the following proteases, respectively: tobacco etch virus protease (TEVp), turnip mosaic virus protease (TUMVp), tobacco vein mottle virus protease (TVMVp), sunflower mild mosaic virus protease (SuMMVp), soybean mosaic virus protease (SbMVp) and plum pox virus protease (PPVp). The artificial signal input cells induce the heterodimerization of the heterodimerization domains, thereby repolymerizing the split protease domains into active complete protease structures, restoring the proteolytic activity of the protease and thus opening the downstream signal process. The virus protease is endonuclease and has accurate amino acid recognition and cutting sequence. The corresponding protease recognition cleavage sequence is introduced into the target protease substrate to form an artificial protease substrate, and the artificial protease substrate is matched with the corresponding protease to realize a specific intracellular signal control effect.
The existing protease tools capable of responding to artificial signal regulation are realized on the basis of the technical route of "split proteins". This means that these regulatable protease tools must be activated in heterodimeric form. In constructing such protease tools, it is necessary to construct and optimize the two-part protease domain separately after cleavage, and in subsequent use, it is necessary to simultaneously transfect the heterologous two-part protease element into mammalian cells. Therefore, the existing controllable protease technical means have certain limitations: one is that the fact that the protease must be heterodimerized after cleavage limits the use of highly efficient homodimeric protein domains for the construction of protease tools; secondly, each protease tool is constructed, two protein domains are required to be constructed, optimized and transfected simultaneously, and the workload of the construction process and the effective packaging capacity occupied on virus transfection vectors are increased.
Based on this situation, the present invention uses a technological route based on mutant natural homodimeric proteases to develop a homodimeric protease tool that can be controlled manually, and at the same time, to develop a series of protease substrates that can be used in combination with the novel proteases to achieve different regulatory functions in cells.
Disclosure of Invention
The invention aims to solve the problem that the existing controllable protease technical means have certain limitation.
To this end, the present invention provides a light-regulated protease tool comprising an HIV-1 protease mutant and a Vivid protein; the Vivid protein is linked to the HIV-1 protease mutant by a flexible peptide; the HIV-1 protease mutant is obtained by mutating an amino acid site which mediates dimerization of HIV-1 protease under natural conditions. The protease is activated by dimerization after being induced by blue light irradiation with the wavelength of 450 nm.
Furthermore, in order to reduce homodimerization activity of the natural HIV-1 protease and ensure that the mutant protease can restore catalytic activity under dimerization mediated by the connected photoinduced dimerization domain, the HIV-1 protease mutant is a T96A mutant or an N98D mutant; wherein the T96A mutant is a mutant which mutates threonine at the 96 th site of HIV-1 protease into alanine; the N98D mutant is a mutant of mutating asparagine at the 98 th position of HIV-1 protease into aspartic acid.
Furthermore, because the homodimerization affinity of the native Vivid protein is low, the construction of the tool for light-regulated protease by directly using the Vivid protein cannot obtain a good protease activation effect, and thus, further mutation screening of the domain of the Vivid protein in the tool for light-regulated protease is required. Preferably, a Vivid protein I52C mutant mutated from isoleucine 52 to cysteine is used as the blue light-responsive homodimerization domain for the construction of a light-regulated protease tool.
Further, the amino acid sequence of the flexible peptide fragment is SGGGSGGGSGGG.
Further, when the Vivid protein mutant is connected to the most C-terminal of the HIV-1 protease mutant, the protease tool shows better post-induction activity, so that the Vivid protein mutant is preferably connected to the C-terminal of the HIV-1 protease mutant through a flexible peptide segment.
In order to realize the control of the protease tool on the intracellular signal process, the invention also provides a matched substrate of the protease tool, wherein the matched substrate comprises a Ub (R) -dK degradation tag, a protease tool recognition cleavage site, a target effector protein, a protease tool recognition cleavage site and 4 serially connected PEST motifs which are sequentially connected from the N end to the C end.
The invention also provides a matched substrate of the protease tool, which comprises a substrate protein, wherein the protease tool recognition cleavage site is inserted into a natural protease recognition cleavage site of the substrate protein; the substrate protein is a protein which is naturally activated after being hydrolyzed by protease.
The invention also provides a matched substrate of the protease tool, which comprises a target effector protein, a protease tool recognition cleavage site and a CAAX sequence which are sequentially connected from the N end to the C end; wherein the CAAX sequence is a CAAX amino acid motif from the end of the C-terminus of the RAS2 protein.
Specifically, because the HIV-1 protease has a plurality of different recognition cleavage sites, in order to realize higher hydrolysis efficiency of the protease on the substrate, the invention optimally tests the recognition sequence of the HIV-1 protease used in the substrate construction process, and selects the optimal recognition cleavage site, and the amino acid sequence of the recognition cleavage site of the protease tool is VSFNFPQITL.
Specifically, the target effector protein is one of fluorescent protein, transcription factor with downstream transcription regulation activity or protein kinase with interference to cell endogenous signal transduction path.
Specifically, the substrate protein is Caspase3 protein or GASDERMIN D protein.
Compared with the prior art, the invention has the following advantages and beneficial effects:
The protease tool which is controlled by the light provided by the invention changes the protease tool which can be controlled by an artificial signal from a heterodimer form to a homodimer form, so that the number of protease tool elements which are required to be transferred into cells in the process of cell experiments is reduced from two to one, and the amino acid sequence length of the protease tool is shortened. By comparing the currently most commonly used heterodimeric protease tools developed based on tobacco etch virus protease (TEVp), the complete light-induced TEVp protease tool is composed of two parts, an N-terminal part TEV protease linked to FKBP protein and a C-terminal part TEV protease linked to Frb protein, the amino acid numbers of which are 244 amino acids and 230 amino acids, respectively, so that the complete use of the TEV protease tool requires transfection of two parts, a total of 474 amino acid-sized proteins. The homodimer protease tool constructed in the invention only needs to be transfected into a structural domain, and the size of the light regulation type protease tool is only 262 amino acids. The more compact structure and smaller protein size allow the protease tool to occupy less of the available packaging capacity of the vector when used, and the protease tool provided by the invention will be more advantageous in some complex artificial signal pathway construction works requiring the transfection of multiple protein elements into cells. When the protease tool provided by the invention is used together with a plurality of different specific substrates matched with the protease tool, the control of the artificial signal input on the signal path in the mammalian cells can be realized through blue light with the wavelength of 450nm to regulate and control the stability of fluorescent protein in the cells, the subcellular localization change of the fluorescent protein, the downstream target gene expression started by a specific transcription factor, apoptosis, cell scorch and other different intracellular signal processes.
The present invention will be described in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a flow chart of screening for combinations of different degradation factors according to example 1 of the present invention.
FIG. 2 is a schematic representation of the protein sequence of a fluorescent protein reporter substrate constructed in example 1 of this invention.
FIG. 3 is a flow chart showing the construction of protease tools for screening and light-regulated protease mutants of HIV-1 in example 1 of the present invention.
FIG. 4 is a schematic representation of the protein sequence organization of the light-regulated protease tools of example 1 of the present invention.
FIG. 5 is a schematic representation of a kit substrate designed based on a protein that is naturally activated after hydrolysis by a protease in example 1 of the present invention.
FIG. 6 is a schematic representation of a supporting substrate designed based on the "CAAX" amino acid motif from the end of the C-terminus of RAS2 protein in example 1 of the present invention.
Reference numerals: 1. ub (R) -dK degradation tag; 2. recognition cleavage sites for protease tools; 3. a target effector protein; 4. PEST motif; 5. vivid protein I52C mutants; 6 flexible peptide segments; 7. HIV-1 protease mutants; 8. CAAX amino acid motif.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in the following examples, and it is obvious that the described examples are only some examples of the present invention, but not all examples. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which the invention pertains will appreciate that various modifications and changes can be made without departing from the scope of the invention. Accordingly, the scope of the invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The technical scheme of the invention specifically comprises two major parts, namely a homodimerization light-induced dimerization activated light-regulated protease tool and a series of matched protease substrates which can be hydrolyzed by the protease tool to generate state change. The substrate in the invention is three types, namely a substrate which is converted into a stable state from a degradable state after being hydrolyzed by protease, a substrate which is converted into a cytoplasmic matrix from a cytoplasmic membrane after being hydrolyzed by protease, and a cell fate control substrate which is activated to induce apoptosis or apoptosis after being hydrolyzed by protease.
The effect of the light-regulated protease tool and its associated substrate according to the invention is examined by means of the following examples.
Example 1:
Among the many natural viral proteases, the HIV-1 (HIV-1 protease) is a homodimeric protease, which is produced by homodimerization of two identical subunits to form a complete catalytic center, and the natural structure meets the requirement of building a homodimeric protease tool, and the embodiment is based on the development of an artificially controllable protease molecular tool of the HIV-1 protease.
1. Construction of a fluorescent protein reporter substrate that can be used to reflect the intracellular Activity of HIV-1 protease
In order to reflect the level of hydrolytic activity in mammalian cells in the subsequent protease tool construction procedure, it is first necessary to construct a reporter substrate that exhibits HIV-1 protease activity in mammalian cells. Development of such substrates based on fluorescent proteins would facilitate quantitative analysis of signals generated by living cells using flow cytometry or fluorescence microscopy imaging techniques. Therefore, in this example, a fluorescent protein design with a degradation tag is used, and a degradation factor is added to the N-terminal or/and the C-terminal of the fluorescent protein and connected by an HIV-1 protease cleavage site, when the HIV-1 protease is inactive, the degradation factor guides the fluorescent protein to be degraded, and when the HIV-1 protease is active, the degradation factors at both ends of the fluorescent protein substrate can be excised, so that the fluorescent protein can exist stably in cells. In order to obtain the best report effect, the present embodiment screens different degradation factor combinations, and the screening flow is shown in fig. 1.
Experimental results show that the N-terminal Ub (R) -dK degradation label and the C-terminal 4 serial PEST motif combination derived from IκB have the largest difference in the activity proportion of cellular fluorescent proteins before and after the hydrolysis of HIV-1 protease, so that the degradation factor combination is selected as a substrate design basis for converting a readily degradable state into a stable state after the hydrolysis by a protease tool, and the protein sequence constitution of a fluorescent protein report substrate is shown in figure 2.
The fluorescent protein report substrate is a substrate which is converted into a stable state from a degradable state after being hydrolyzed by protease tools, and target effector proteins in the substrate can be the fluorescent protein with the report function, transcription factors with downstream transcription regulation activity, such as Tet3G, gal4 and the like, and protein kinase with interference effect on cell endogenous signal transduction pathways. The regulation and control function which can be realized according to the needs in actual use can replace the required target effector protein based on the substrate design principle provided by the invention, and the process of controlling the conversion of the target effector protein in cells from the closed state to the open state by a protease tool is realized.
2. Connecting HIV-1 protease mutants with photoinduced dimerization domain for HIV-1 protease mutant screening to construct a light-regulated protease tool
In the complete HIV-1 protease structure, the N-terminal and C-terminal amino acids of the two subunits form four antiparallel, interdigitated beta-sheet structures, which form the terminal dimerization plane of the HIV-1 protease, so that the N-terminal and C-terminal subunits are key regions that determine the spontaneous dimerization activity of the HIV-1 protease subunits under natural conditions. In order to construct proteases that are regulated by artificial signal input, it is necessary to mutate or combine mutations at key amino acid sites that mediate dimerization of HIV-1 protease under natural conditions, thereby disrupting the natural dimerization of HIV-1 protease and disrupting the natural hydrolytic activity of HIV-1 protease. For this purpose, 12 HIV-1 protease mutants were screened in this example as shown in Table 1.
Table 1 12 mutants of HIV-1 protease screened during construction of protease tools
| Mutation name | Mutation position | Original amino acid | Post-mutation amino acids |
| D25N | 25 | Aspartic acid (D) | Asparagine (N) |
| P1A | 1 | Proline (P) | Alanine (A) |
| Q2A | 2 | Glutamine (Q) | Alanine (A) |
| I3A | 3 | Isoleucine (I) | Alanine (A) |
| T96A | 96 | Threonine (T) | Alanine (A) |
| L97A | 97 | Leucine (L) | Alanine (A) |
| N98D | 98 | Asparagine (N) | Aspartic acid (D) |
| F99A | 99 | Phenylalanine (F) | Alanine (A) |
| 97&99A | 97 And 99 | Leucine and phenylalanine | Alanine (A) |
| Δ99 | 99 | Phenylalanine (F) | Truncating |
| I3A&Δ99 | 3 And 99 | Isoleucine and phenylalanine | Alanine at position 3, truncate 99 |
The photo-induced homodimeric domain was derived from the Vivid protein in neurospora crassa. Because the homodimerization affinity of the natural Vivid protein is low, the construction of the light-regulated protease tool by directly using the Vivid protein cannot obtain a good protease activation effect, so that further mutation screening of the Vivid protein domain in the light-regulated protease tool is required. The mutation of the key site affecting the dimerization affinity of the Vivid protein was performed, and according to the results of experimental tests, the final example used the Vivid protein mutant (I52C) with isoleucine at position 52 mutated to cysteine as the blue light-responsive homodimerization domain for the construction of a light-regulated protease tool.
As shown in FIG. 3, the mutated HIV-1 protease mutant is connected with a Vivid protein I52C mutant induced by 450nm blue light through a flexible peptide segment, so that a protease tool to be screened and regulated by light is constructed, and the amino acid sequence connected with the flexible peptide segment is SGGGSGGGSGGG. The protease tools to be screened were co-transfected into a human embryonic kidney cell line (HEK-293T cell line) with the above fluorescent protein reporter substrate and the effect of protease tools with different HIV-1 protease mutants on the substrate hydrolytic activity before and after dimerization using blue light of 450nm wavelength induced Vivid protein I52C mutants was analyzed by flow cytometry.
In order to reduce the homodimerization activity of the natural HIV-1 protease and ensure that the mutant protease can restore the catalytic activity under the dimerization mediated by the connected photoinduced dimerization domain. According to the screening result of the HIV-1 protease mutants, the T96A mutant and the N98D mutant are determined to be the optimal HIV-1 protease mutants for constructing protease tools. Wherein the T96A mutant is a mutant in which threonine at position 96 of HIV-1 protease is mutated to alanine, and the N98D mutant is a mutant in which asparagine at position 98 of HIV-1 protease is mutated to aspartic acid.
In order to make the light-regulated protease tool have stronger activation effect, the Vivid protein I52C mutant is connected to the C end of the HIV-1 protease mutant during construction. The protein sequence composition of the light-regulated protease tool is shown in FIG. 4.
3. Matched substrate of protease tool controlled by light
In addition to the above fluorescent protein reporter substrates, this example also developed two types of support substrates for light-regulated protease tools.
1. Based on proteins activated by natural proteolytic enzymes
The protein activated by the protease after the hydrolysis mainly exists in the signal process of apoptosis, cell pyrosis and the like, and in general, the type of protein has two subunits with effects and inhibiting effects, and a specific protease hydrolysis cleavage site is arranged between the two subunits. When hydrolyzed by an upstream protease, the inhibitory subunit separates from the effector subunit, releasing the inhibitory effect, and the protein produces a downstream effect.
Thus, insertion of the cleavage recognition site of the protease tool (HIV-1 protease cleavage site) into the natural protease recognition cleavage site of the substrate protein allows the substrate to be activated under the control of an artificially designed protease tool while disrupting the natural protease recognition cleavage site of the substrate. This example contains two types of support substrates of this type, the structure of which is shown in FIG. 5.
2. Based on the "CAAX" amino acid motif from the end of the C-terminal of the RAS2 protein
Proteins containing the "CAAX" amino acid motif from the end of the RAS2 protein at the C-terminus are recognized in mammalian cells after translation by farnesyl transferase and myrcenyl transferase, resulting in prenylation modifications from the cysteine site on the motif, and the prenylated modified proteins are anchored to the plasma membrane. In order to realize the regulation and control of protease tools on subcellular localization of target proteins in cells, the design of the matched substrates is as follows: a "CAAX" amino acid motif is added to the C-terminus of a particular target protein and is linked between the motif and the original protein by a recognition cleavage site for protease means (HIV-1 protease cleavage site). The protein substrate thus designed will localize to the cell membrane when the protease means is not activated, whereas subcellular localization of the target substrate released from the cell membrane to the cytoplasm or nucleus (if the target protein contains a nuclear localization sequence) can be achieved by manually controlling activation of the protease means. A schematic of the design of such a substrate is shown in FIG. 6.
4. Recognition cleavage site optimization of protease tools
Because HIV-1 protease has a plurality of different recognition cleavage sites, in order to realize higher hydrolysis efficiency of the protease on the substrate, the HIV-1 protease recognition sequence used in the substrate construction process is optimally tested in the embodiment, and the optimal recognition cleavage site is selected, and the amino acid sequence of the recognition cleavage site is VSFNFPQITL.
The foregoing examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and all designs that are the same or similar to the present invention are within the scope of the present invention.
Claims (3)
1. A protease tool and support substrate, characterized by: the protease tools include HIV-1 protease mutants and Vivid proteins; the Vivid protein is linked to the HIV-1 protease mutant by a flexible peptide; the HIV-1 protease mutant is obtained by mutating an amino acid site mediating HIV-1 protease dimerization under natural conditions;
The HIV-1 protease mutant is an N98D mutant; wherein the N98D mutant is a mutant which mutates asparagine at the 98 th position of HIV-1 protease into aspartic acid;
The Vivid protein is a Vivid protein I52C mutant; the Vivid protein I52C mutant is a mutant which mutates the 52 th isoleucine of the Vivid protein into cysteine;
the Vivid protein domain is connected to the C end of the HIV-1 protease mutant through a flexible peptide;
The amino acid sequence of the flexible peptide fragment is SGGGSGGGSGGG;
The matched substrate comprises a Ub (R) -dK degradation tag, a protease tool recognition cleavage site, a target effector protein, a protease tool recognition cleavage site and 4 serially connected PEST motifs which are sequentially connected from an N end to a C end;
the amino acid sequence of the protease tool recognition cleavage site is VSFNFPQITL;
The target effector protein is one of fluorescent protein, transcription factor with downstream transcription regulation activity or protein kinase with interference to cell endogenous signal transduction path.
2. A protease tool and support substrate, characterized by: the protease tools include HIV-1 protease mutants and Vivid proteins; the Vivid protein is linked to the HIV-1 protease mutant by a flexible peptide; the HIV-1 protease mutant is obtained by mutating an amino acid site mediating HIV-1 protease dimerization under natural conditions;
The HIV-1 protease mutant is an N98D mutant; wherein the N98D mutant is a mutant which mutates asparagine at the 98 th position of HIV-1 protease into aspartic acid;
The Vivid protein is a Vivid protein I52C mutant; the Vivid protein I52C mutant is a mutant which mutates the 52 th isoleucine of the Vivid protein into cysteine;
the Vivid protein domain is connected to the C end of the HIV-1 protease mutant through a flexible peptide;
The amino acid sequence of the flexible peptide fragment is SGGGSGGGSGGG;
The matched substrate comprises a substrate protein, and a protease tool recognition cleavage site is inserted into a natural protease recognition cleavage site of the substrate protein; the substrate protein is a protein which is naturally activated after being hydrolyzed by protease;
the amino acid sequence of the protease tool recognition cleavage site is VSFNFPQITL;
the substrate protein is Caspase3 protein or GASDERMIN D protein.
3. A protease tool and support substrate, characterized by: the protease tools include HIV-1 protease mutants and Vivid proteins; the Vivid protein is linked to the HIV-1 protease mutant by a flexible peptide; the HIV-1 protease mutant is obtained by mutating an amino acid site mediating HIV-1 protease dimerization under natural conditions;
The HIV-1 protease mutant is an N98D mutant; wherein the N98D mutant is a mutant which mutates asparagine at the 98 th position of HIV-1 protease into aspartic acid;
The Vivid protein is a Vivid protein I52C mutant; the Vivid protein I52C mutant is a mutant which mutates the 52 th isoleucine of the Vivid protein into cysteine;
the Vivid protein domain is connected to the C end of the HIV-1 protease mutant through a flexible peptide;
The amino acid sequence of the flexible peptide fragment is SGGGSGGGSGGG;
the matched substrate comprises a target effector protein, a protease tool recognition cleavage site and a CAAX sequence which are sequentially connected from an N end to a C end; wherein the CAAX sequence is a CAAX amino acid motif from the end of the C-terminus of the RAS2 protein;
the amino acid sequence of the protease tool recognition cleavage site is VSFNFPQITL;
The target effector protein is one of fluorescent protein, transcription factor with downstream transcription regulation activity or protein kinase with interference to cell endogenous signal transduction path.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007053792A2 (en) * | 2005-11-05 | 2007-05-10 | Amplyx Pharmaceuticals, Inc. | Improving the pharmacokinetics of protease inhibitors and other drugs |
| WO2018069782A2 (en) * | 2016-10-12 | 2018-04-19 | Kemijski Institut | A combination of split orthogonal proteases with dimerization domains that allow for assembly |
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| US9163094B2 (en) * | 2011-09-27 | 2015-10-20 | Massachusetts Institute Of Technology | Light-activated fusion proteins and uses therefor |
| US20160326219A1 (en) * | 2013-12-13 | 2016-11-10 | Ist Austria (Institute Of Science And Technology Austria) | Optically activated receptors |
| US11965002B2 (en) * | 2018-11-29 | 2024-04-23 | The Bd Of Trustees Of The University Of Illinois | Optogenetic construct for allosteric control of protein activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007053792A2 (en) * | 2005-11-05 | 2007-05-10 | Amplyx Pharmaceuticals, Inc. | Improving the pharmacokinetics of protease inhibitors and other drugs |
| WO2018069782A2 (en) * | 2016-10-12 | 2018-04-19 | Kemijski Institut | A combination of split orthogonal proteases with dimerization domains that allow for assembly |
Non-Patent Citations (2)
| Title |
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| Genetically engineered photoinducible homodimerization system with improved dimer-forming efficiency;Yuta Nihongaki等;ACS Chem Biol;20140321;第9卷(第3期);全文 * |
| Mutagenesis of the dimer interface residues of tethered and untethered HIV-1 protease result in differential activity and suggest multiple mechanisms of compensation;Sumana Choudhury等;Virology;20030315;第307卷(第2期);全文 * |
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