A modified KIFC1 protein, a KIFC1-DARPin D1-tubulin protein complex and its applications

Abstract

This invention relates to the field of biotechnology, specifically to a modified KIFC1 protein, a KIFC1-DARPin D1-tubulin protein complex, and their applications. The invention provides a modified KIFC1 protein, the amino acid sequence of which is shown in SEQ ID NO.2. By recombining the domains of the wild-type KIFC1 protein, the obtained modified KIFC1 protein exhibits high expression levels, good stability, and ease of purification, solving the technical problem of difficult expression and purification of wild-type KIFC1. Based on this, the invention, for the first time, successfully prepared a high-purity, highly uniform KIFC1-DARPin D1-tubulin protein ternary complex by assembling the modified KIFC1 protein with DARPin D1 and tubulin proteins in a specific molar ratio, filling the gap in the prior art regarding the lack of stable KIFC1-related ternary complexes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a modified KIFC1 protein, a KIFC1-DARPin D1-tubulin protein complex, and their applications. Background Technology

[0002] Kinesin family member C1 (KIFC1) is a unique negative-terminal oriented microtubule motor protein belonging to the kinesin-14 family. Microtubules are cytoskeletal structures dynamically assembled from α / β-tubulins, participating in crucial life activities such as intracellular transport, spindle assembly during mitosis, centrosome localization, and chromosome segregation. KIFC1 binds to microtubules through its C-terminal motility domain, utilizing energy from ATP hydrolysis to move along the microtubules towards the negative end. During cell division, it mediates centrosome aggregation, spindle assembly, and maintains bipolar spindle stability, which is essential for ensuring accurate chromosome segregation. Studies have shown that KIFC1 is highly expressed in various tumor cells, especially in tumor cells with centrosome expansion. KIFC1 maintains cell survival by assembling multipolar spindles, making it an important potential target for tumor-targeted therapy.

[0003] DARPin (Designed Ankyrin Repeat Protein) is a class of artificially designed binding proteins based on natural ankyrin repeat sequences. It possesses advantages such as high stability, high affinity, and ease of expression and purification, and can specifically recognize and bind to target proteins. DARPin D1 is a typical DARPin molecule targeting β-tubulin, capable of specifically binding to tubulin dimers or microtubule structures. It can be used to regulate microtubule dynamics, label microtubule terminals, or interfere with microtubule-related protein interactions, and has significant application value in microtubule function research and structural biology.

[0004] Currently, studies have reported the interaction mechanism between KIFC1 and tubulin protein, as well as the binding characteristics of DARPin D1 and tubulin protein. For example, KIFC1 binds to soluble tubulin through its N-terminal tail domain, forming a heterogeneous motor-tubulin cluster that drives the formation of the microtubule-negative aster, regulating the transition between microtubule bundles and the aster (https: / / www.nature.com / articles / s41467-021-27528-6). However, no literature has yet reported whether KIFC1, DARPin D1, and tubulin protein can form a stable ternary protein complex. Because the interaction between KIFC1 and tubulin is dynamic and has limited binding affinity, coupled with the poor stability of tubulin itself, it is difficult to obtain a KIFC1-tubulin protein complex with good homogeneity and high stability. If a stable KIFC1-DARPin D1-tubulin ternary complex can be obtained, the high affinity of DARPin D1 can stabilize the binding of KIFC1 and tubulin, providing a high-quality sample for elucidating the structural basis of the interaction between KIFC1 and tubulin. At the same time, it can provide a stable in vitro screening model for anti-tumor drugs based on KIFC1 targets. This is of great significance for a deeper understanding of the molecular mechanism of KIFC1 and the development of anti-tumor drugs targeting KIFC1. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a modified KIFC1 protein, a KIFC1-DARPin D1-tubulin protein complex, and its application.

[0006] The present invention achieves the above objectives through the following technical solutions: The present invention provides a modified KIFC1 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] As a further optimization of the above invention, the modified KIFC1 protein further includes a fusion tag and an enzyme cleavage site, and the amino acid sequence of the modified KIFC1 protein containing the fusion tag and enzyme cleavage site is shown in SEQ ID NO.3.

[0008] As a further optimization of the above invention, the following steps are included: modifying wild-type KIFC1 using gene editing techniques to obtain a gene encoding the modified KIFC1 protein; ligating the gene into an expression vector and transforming it into host cells for induced expression; and purifying the modified KIFC1 protein by affinity chromatography, heparin chromatography, and gel filtration chromatography.

[0009] The present invention provides a polynucleotide that encodes the modified KIFC1 protein as described above, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0010] The present invention provides a recombinant expression vector, wherein the expression vector is pET-28a.

[0011] This invention provides the application of the modified KIFC1 protein in the preparation of the KIFC1-DARPin D1-tubulin protein ternary complex, comprising the following steps: (1) Mix the modified KIFC1 protein, DARPin D1 protein and tubulin protein; (2) Add adenosine-5'-diphosphate (ADP), adenosine-5'-triphosphate (ATP), and adenosine-5'-(β,γ-imido)triphosphate (AMPPNP) to the mixed system and incubate on ice; (3) The KIFC1-DARPin D1-tubulin protein ternary complex was obtained by gel filtration chromatography purification.

[0012] As a further optimization of the above invention, the amino acid sequence of the DARPin D1 protein is shown in SEQ ID NO.5.

[0013] As a further optimization of the above invention, the molar ratio of the modified KIFC1 protein, DARPin D1 protein and tubulin protein is (1.2-1.6):(1.6-2.0):(0.8-1.2).

[0014] This invention provides an application of the KIFC1-DARPin D1-tubulin protein ternary complex as described above in protein structure analysis or antitumor drug screening.

[0015] The beneficial effects of this invention are as follows: By recombining the domains of wild-type KIFC1 protein and replacing amino acids 139-304 with the linker peptide GGSGGSGGSGGS, the resulting recombinant KIFC1 protein exhibits high expression levels, good stability, and ease of purification, solving the technical problem of difficult expression and purification of wild-type KIFC1. Furthermore, this invention, for the first time, successfully prepared a high-purity, highly uniform KIFC1-DARPin D1-tubulin protein ternary complex by assembling the modified KIFC1 protein with DARPin D1 and tubulin proteins in a specific molar ratio. This fills the gap in existing technologies for the lack of stable KIFC1-related ternary complexes, providing important tool proteins and technical support for the study of KIFC1-tubulin interaction mechanisms, protein structure analysis, and anti-tumor drug screening. Attached Figure Description

[0016] Figure 1 Figure showing the results of small-scale purification of KIFC1 protein; Figure 2 Figure showing the results of small-scale purification of DARPin D1 protein; Figure 3 Results of His affinity chromatography and affinity chromatography after enzyme digestion of KIFC1 protein; Figure 4 The results are from the heparin affinity chromatography of KIFC1 protein. Figure 5 Image showing SEC detection of KIFC1 protein; Figure 6 For comparison, use the affinity chromatography results of KIFC1 protein; Figure 7 The results of KIFC1 protein gel filtration chromatography are for reference. Figure 8 Results of His affinity chromatography and affinity chromatography after enzyme digestion for DARPin D1 protein; Figure 9 This is a SEC detection image of DARPin D1 protein; Figure 10 SEC detection chromatogram for KIFC1-DARPin D1-tubulin complex; Figure 11 SEC detection chromatogram prepared for the control KIFC1-DARPin D1-tubulin complex. Detailed Implementation

[0017] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0018] 1. Materials and Reagents Unless otherwise specified, all reagents used in this experiment are conventional reagents and were prepared using deionized water. All instruments used are standard laboratory instruments, and all methods used in this experiment are standard methods in this field unless otherwise specified.

[0019] 2. Method 2.1 Gene Synthesis of KIFC1 and DARPin D1 Based on the amino acid sequence of wild-type KIFC1 (Uniprot number Q9BW19, SEQ ID NO.1), a modified KIFC1 protein was designed by replacing amino acid positions 139-304 with the linker peptide GGSGGSGGSGGS, resulting in the amino acid sequence of the modified KIFC1 protein (SEQ ID NO.2). The target gene sequence was obtained through gene synthesis (SEQ ID NO.4) and cloned into the pET-28a expression vector. A 6His-TEV tag was added to the N-terminus to construct the recombinant plasmid pET-28a-6His-TEV-KIFC1(M1-D138)-GGSGGSGGSGGS-KIFC1(Q305-K673). Here, "6His" is the affinity purification tag, and "TEV" is the protease cleavage site used for subsequent tag removal, yielding the amino acid sequence of the modified KIFC1 protein with the fusion tag (SEQ ID NO.3). Simultaneously, the DARPin D1 gene was synthesized, with its amino acid sequence shown in SEQ ID NO.5, and constructed into the same expression vector and with the same fusion tag as KIFC1. All synthesized genes have been verified as correct by sequencing companies.

[0020] Simultaneously, a previously reported KIFC1 protein was constructed as a control (hereinafter referred to as the KIFC1 control protein). This control protein was derived from PDB ID 5WDH, namely pFastBac1-6His-Thrombin-KIFC1 (L307-C663) (T368P). Here, "6His" is the affinity purification tag, and "Thrombin" is the protease cleavage site used for subsequent tag removal. The synthesized genes were verified by a sequencing company. The coding sequence of the control KIFC1 protein with the fusion tag is shown in SEQ ID NO. 6.

[0021] 2.2 KIFC1 and DARPin D1 Small-Scale Expression Test 2.2.1 Low-level expression of KIFC1 The correctly sequenced KIFC1 recombinant plasmid was transformed into T7 express competent cells and cultured overnight at 37°C. Single colonies were picked and inoculated into 5 mL LB broth and cultured at 37°C until the bacterial culture reached OD500. 600 When the pH is 0.6-0.8, a small amount of bacterial culture is fixed with loading buffer, and another small amount is added to glycerol and stored at -80℃ for later use. The remaining bacterial culture is added to 0.5 mM IPTG and induced at 16℃ for 12 hours. The bacterial cells are collected, and the induced bacterial culture is analyzed by SDS-PAGE. The results are as follows: Figure 1 As shown, a clear target protein band is visible.

[0022] 2.2.2. Small-scale expression and purification of DARPin D1 The correctly sequenced DARPin D1 recombinant plasmid was transformed into BL21(DE3) competent cells and cultured overnight at 37°C. Single colonies were picked and inoculated into 5 mL LB liquid medium and cultured at 37°C until the bacterial culture reached OD500. 600 When the pH is 0.6-0.8, a small amount of bacterial culture is fixed with loading buffer, and another small amount is added to glycerol and stored at -80℃ for later use. The remaining bacterial culture is added to 0.5 mM IPTG and induced at 15℃ for 16 hours. The bacterial cells are collected, and the induced bacterial culture is analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown, a clear target protein band is visible.

[0023] 2.3 Large-scale expression and purification of KIFC1 A. Large-scale expression and purification of modified KIFC1 The KIFC1 strain identified as positive in step 2.2.1 was inoculated into 50 mL of LB liquid medium and cultured overnight at 37°C. It was then transferred to 1 L of LB liquid medium at a 1:100 ratio and cultured at 37°C until the OD600 reached 0.6-0.8. 0.5 mMIPTG was then added, and the culture was induced at 16°C for 12 hours. The bacterial cells were collected by centrifugation at 5000 rpm for subsequent purification.

[0024] The purification steps are as follows: (1) Lysis and centrifugation: Weigh the collected bacterial cells and add lysis buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 1 mM PMSF) at a ratio of 1:10. Use a high-pressure homogenizer to break the bacterial cells, centrifuge at 16000 rpm, and collect the supernatant.

[0025] (2) Affinity chromatography: Purification was performed using a Ni Bestarose FF affinity chromatography column, utilizing the His tag at the N-terminus of the KIFC1 protein. The column was first equilibrated to 10 column volumes with washing buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol). The lysate supernatant was loaded onto the column, and the target protein was eluted with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol, 500 mM imidazole). The eluent was collected, and the results are shown below. Figure 3 As shown.

[0026] (3) Tag removal: Add TEV enzyme to the elution buffer and digest overnight at 4°C to remove the N-terminal 6His tag. The digested product is loaded again onto a Ni Bestarose FF affinity chromatography column. The untagged KIFC1 protein permeates out, and the permeate is collected.

[0027] (4) Heparin affinity chromatography: The permeate was diluted with dilution buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 5% glycerol) and loaded onto a pre-equilibrated HiTrap Heparin HP heparin affinity chromatography column. A linear gradient elution was used, with the elution buffer (50 mM Tris-HCl pH 8.0, 1000 mM NaCl, 5% glycerol) ratio increasing from 0% to 100%, and the gradient duration being 10-20 column volumes. The eluted fraction was collected based on the UV absorption peak and analyzed by SDS-PAGE (e.g., ...). Figure 4 As shown in the figure, the target protein Part 2 was collected.

[0028] (5) Gel filtration chromatography: The collected Part 2 eluent was concentrated and subjected to Superdex 200 Increase 10 / 300 GL gel filtration chromatography. The SEC buffer was 20 mM Tris-HCl pH 8.0, 5% glycerol, and 250 mM NaCl. The target protein (e.g., Figure 5 (As shown).

[0029] B. Large-scale expression and purification of KIFC1 control samples The control KIFC1 protein (derived from PDB ID 5WDH, amino acid L307-C663, containing the T368P mutation) was expressed using an insect cell-baculovirus expression system. The specific steps are as follows: The recombinant plasmid pFastBac1-6×His-Thrombin-KIFC1(L307-C663) (T368P) was transformed into DH10Bac competent cells (Bomaide, BC112-01) and cultured at 37°C for 48 hours. Positive clones were obtained by blue-white screening, and positive clones were picked and cultured overnight in LB medium. Recombinant Bacmid (baculovirus plasmid) was extracted.

[0030] Insect Sf9 cells (Thermo, 11496015) were diluted to 1×10⁻⁶. 6 Cells / mL. Take 15 μL of recombinant Bacmid and add it to 100 μL of Grace insect medium (Thermo, 10902104) containing 7 μL of X-treme transfection reagent (Roche). Mix well and incubate for 15 minutes. Transfect the mixture into Sf9 cells, and harvest the P0 generation recombinant baculovirus after 4 days of culture. Add 1 mL of P0 virus to 50 mL of Sf9 cells, culture for 3 days, centrifuge for 5 minutes, and collect the supernatant as P1 virus. Add 4 mL of P1 virus to 200 mL of Sf9 cells, culture for 3 days, centrifuge for 5 minutes, and collect the supernatant as P2 virus.

[0031] The prepared P2 generation virus was transfected into Hi-5 cells (Thermo, B85502) at a ratio of 3 μL / 0.5 mL, with a total expression volume of 1.6 L. After culturing at 27°C for 48 hours, the cells were collected for subsequent purification.

[0032] The purification steps are as follows: Weigh the collected bacterial cells and add lysis buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 1 mM PMSF, 2 mM β-ME) at a 1:10 ratio. Homogenize by high pressure and collect the supernatant by centrifugation at 16000 rpm. Purify using a His FF affinity chromatography column with an N-terminal His tag. First, equilibrate the column to 10 column volumes with washing buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 2 mM β-ME). Load the lysis supernatant onto the column and elute the target protein with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 2 mM β-ME, 500 mM imidazole). The results are shown below. Figure 6 As shown, KIFC1 was successfully eluted.

[0033] The collected eluent was concentrated and subjected to gel filtration chromatography using a HiLoad 16 / 600 Superdex 200 pg column. The SEC buffer consisted of 20 mM Tris-HCl pH 8.0, 5% glycerol, 150 mM NaCl, and 2 mM DTT. Peak 1 (the target protein) was collected based on the peak chromatogram. Figure 7 ).

[0034] 2.4. Large-scale expression and purification of DARPin D1 The DARPin D1 strain identified as positive in step 2.2.2 was inoculated into 50 mL of LB liquid medium and incubated overnight at 37°C. It was then transferred to 1 L of LB liquid medium at a 1:100 ratio and incubated at 37°C until OD (digestion / degradation). 600 When the concentration is 0.6-0.8, add 0.5 mMIPTG and induce overnight at 15°C. Collect the cells by centrifugation at 5000 rpm for subsequent purification.

[0035] The purification steps are as follows: (1) Lysis and centrifugation: Weigh the collected bacterial cells and add lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 1% NP-40, 5 mM MgCl2, 1 mM ATP) at a ratio of 1:10. Use a high-pressure homogenizer to break the bacterial cells, centrifuge at 16000 rpm, and collect the supernatant.

[0036] (2) Affinity chromatography: Purification was performed using a His FF affinity chromatography column, utilizing the His tag at the N-terminus of the DARPin D1 protein. The column was first equilibrated to 10 column volumes with washing buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM MgCl2, 1 mM ATP). The lysate supernatant was loaded onto the column, and the target protein was eluted with elution buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM MgCl2, 1 mM ATP, 300 mM imidazole). The eluent was collected, and the results are shown below. Figure 8 As shown.

[0037] (3) Tag removal: TEV enzyme was added to the elution buffer and the mixture was digested overnight at 4°C to remove the N-terminal 6His tag. The digested product was then loaded back onto a Talon affinity chromatography column. The untagged DARPin D1 protein permeated through the column, and the permeate was collected. The results are as follows: Figure 8 As shown, protein samples were taken for SDS-PAGE analysis at each step.

[0038] (4) Gel filtration chromatography: The collected permeate was concentrated and subjected to Superdex 75 Increase 10 / 300 GL gel filtration chromatography. The SEC buffer was 20 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM MgCl2, 1 mM MEGTA, and 1 mM DTT. The target protein peak Peak 3 (e.g., peak 3) was collected according to the peak diagram. Figure 9 (As shown).

[0039] 2.5 Preparation of the KIFC1-DARPin D1-tubulin protein ternary complex A. Preparation of the modified KIFC1-DARPin D1-tubulin protein ternary complex The modified KIFC1 protein purified in step 2.3, the DARPin D1 protein purified in step 2.4, and commercial tubulin protein (purchased from Shanghai Unimicron Biotechnology Co., Ltd., Cat. No. T240-B) were mixed at a molar ratio of 1.4:1.8:1, and 1 mM ADP, 1 mM ATP, and 1 mM AMPPNP were added. The mixture was incubated on ice for 40 minutes. The tubulin protein consists of tubulin α and tubulin β. The amino acid sequence of tubulin α is shown in SEQ ID NO.7, and the amino acid sequence of tubulin β is shown in SEQ ID NO.8.

[0040] The above mixture was subjected to Superdex 200 Increase 10 / 300 GL gel filtration chromatography. The SEC buffer consisted of 20 mM PIPES-KOH pH 6.8, 50 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, and 10 μM AMPPNP. The target protein was collected based on the gel filtration chromatography peak chromatogram. Figure 10 Peak1 is the target protein. The protein has high purity and the peak shape is symmetrical and sharp, indicating that the complex has good homogeneity in solution.

[0041] Preparation of the KIFC1 control sample DARPin D1-tubulin protein ternary complex Following the same protocol as in section A, complexes were prepared from control KIFC1, DARPin D1, and tubulin samples. The gel chromatography results are shown below. Figure 11 The control KIFC1 did not form a complex with Tubulin and did not bind to DARPin D1, indicating that the control KIFC1 did not form a stable complex with DARPin D1-tubulin.

[0042] 3. Conclusion By recombining the domains of wild-type KIFC1 protein, replacing amino acids 139-304 with the linker peptide GGSGGSGGSGGS, the resulting recombinant KIFC1 protein (SEQ ID NO.2) exhibits high expression levels, good stability, and ease of purification, effectively solving the technical problem of difficult expression and purification of wild-type KIFC1. Based on this, this invention, for the first time, successfully prepared a high-purity, highly homogeneous KIFC1-DARPin D1-tubulin protein ternary complex by mixing the modified KIFC1 protein with DARPin D1 and tubulin proteins at a molar ratio of 1.4:1.8:1 and followed by gel filtration chromatography. Figure 10 SDS-PAGE and SEC results showed that the complex had high purity, sharp and symmetrical peaks, and good homogeneity in solution. This invention solves the technical problem of obtaining stable KIFC1-related ternary complexes in the prior art, providing important tool proteins and technical support for the study of KIFC1-tubulin interaction mechanisms, protein structure analysis, and anti-tumor drug screening, and has significant scientific research value and application prospects.

[0043] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An engineered KIFC1 protein, characterized in that, The amino acid sequence of the modified KIFC1 protein is shown in SEQ ID NO.

2.

2. The modified KIFC1 protein according to claim 1, characterized in that, The modified KIFC1 protein further includes a fusion tag and an enzyme cleavage site, and the amino acid sequence of the modified KIFC1 protein containing the fusion tag and enzyme cleavage site is shown in SEQ ID NO.

3.

3. A method for preparing the modified KIFC1 protein as described in any one of claims 1-2, characterized in that, Includes the following steps: Wild-type KIFC1 was modified using gene editing techniques to obtain a gene encoding the modified KIFC1 protein; the gene was ligated into an expression vector and transformed into host cells for induced expression; the modified KIFC1 protein was obtained by affinity chromatography, heparin chromatography, and gel filtration chromatography.

4. A polynucleotide, characterized in that, The polynucleotide encodes the modified KIFC1 protein as described in claim 2, and its nucleotide sequence is shown in SEQ ID NO.

4.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide of claim 4.

6. The recombinant expression vector according to claim 5, characterized in that, The expression vector is pET-28a.

7. The use of the modified KIFC1 protein as described in any one of claims 1-2 in the preparation of the KIFC1-DARPin D1-tubulin protein ternary complex, characterized in that, Includes the following steps: (1) Mix the modified KIFC1 protein, DARPin D1 protein and tubulin protein; (2) Add ADP, ATP and AMPPNP to the mixture and incubate on ice; (3) The KIFC1-DARPin D1-tubulin protein ternary complex was obtained by gel filtration chromatography purification.

8. The application according to claim 7, characterized in that, The amino acid sequence of DARPin D1 protein is shown in SEQ ID NO. 5; the tubulin protein is a heterodimer formed by tubulin α and tubulin β, the amino acid sequence of tubulin α is shown in SEQ ID NO. 7, and the amino acid sequence of tubulin β is shown in SEQ ID NO.

8.

9. The application according to claim 7, characterized in that, The molar ratio of the modified KIFC1 protein, DARPin D1 protein, and tubulin protein is (1.2-1.6):(1.6-2.0):(0.8-1.2).

10. The application of the KIFC1-DARPin D1-tubulin protein ternary complex as described in claim 7 in protein structure analysis or antitumor drug screening.

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