CN112480245B - Application of hydrophobic cyclic peptide ligand in purification of human immunoglobulin G - Google Patents
Application of hydrophobic cyclic peptide ligand in purification of human immunoglobulin G Download PDFInfo
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Abstract
The invention relates to an application of a hydrophobic cyclic peptide ligand for purifying human immunoglobulin G, wherein the sequence of the hydrophobic cyclic peptide ligand is- [ HWG-C-AKTE]‑、‑[YF‑C‑WRHE]‑、‑[WV‑C‑LHHYF]‑、‑[PYF‑C‑TIE]‑、‑[FY‑C‑DEHL]The hydrophobic cyclic peptide ligand is obtained by computer simulation of a biomimetic design using SpA and C on hIgG-Fc fragment H The amino acid sequence Ser383-Asn389 on the binding region 3 is a simulation system, hydrophobic cyclic peptide ligand which can have specific binding effect with hIgG is determined through LeDock molecular docking and FlexX molecular docking and Amber molecular dynamics simulation screening, and the cyclic peptide ligand primary chromatography medium is adopted to carry out purification research on the hIgG from human serum samples, so that the purity of the hIgG can reach 96% and the recovery rate of 92%.
Description
Technical Field
The invention relates to the technical field of purification of human immunoglobulin G, which adopts a computer simulation technology to simulate and design an affinity peptide ligand library of human IgG, and then utilizes an affinity chromatography test to verify the purification effect of peptide molecules on the human IgG, and belongs to the technical field of computer simulation and downstream protein separation and purification in bionics design.
Background
Currently, antibodies are widely used in medical diagnosis and disease treatment, and more than 20 antibody classes of drugs approved by the FDA in 2006 are sold worldwide for over 170 billion dollars, wherein IgG is the main antibody component in serum, accounting for about 75% of serum Ig, and is the most demanding class of antibodies. The preparation of antibody medicine is usually completed by precipitation, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and other methods, and the process cost is about 30-40% of the production cost. Staphylococcus aureus protein A (SpA), protein G and protein L are widely used as affinity ligands for preparing high-purity antibodies, the ligand has high specificity, however, too high affinity requires more severe elution conditions, denaturation of target proteins and ligand shedding are easy to cause, the adsorption capacity is low, in addition, the preparation of the ligand is difficult and expensive, and the protein generally loses part of activity after immobilization, so that the application of the protein ligand is limited.
The polypeptide has more superiority as an affinity ligand, but the number of the polypeptide with affinity with target protein in nature is very limited, and the problems of selecting a proper polypeptide sequence as the affinity ligand and improving the affinity and selectivity of the polypeptide are key to influencing the application of polypeptide affinity chromatography. The existing screening and design methods are mainly divided into experimental screening and rational design, wherein the experimental screening is performed by high-throughput experimental screening based on a combinatorial library technology, and the rational design is mainly performed by designing new ligands based on the structure and properties of target proteins or existing ligands.
The university of Tianjin Sun Yan professor task group uses 6 SpA key residues: f132, Y133, H137, E143, R146 and K154 construct a simulation system, molecular docking and molecular dynamics simulation are utilized to screen a polypeptide library, linear octapeptide molecule FYWHCLDE, FYTHCAKE with the purity of human IgG being more than 88% and the recovery rate being more than 65% can be obtained, although the purification effect is better, the purification effect is also longer than commercial application, the simulation is based on a hydrophobic action model between protein A and IgG, the obtained polypeptide sequence is combined with hIgG based on electrostatic action, the electrostatic action is known in the art not to be specific adsorption action, other biomass molecules are easy to adsorb, the purity of the obtained protein is not high, and the commercial utilization is difficult.
Disclosure of Invention
The invention is based on protein A and C on the hIgG-Fc fragment H 3 binding region (hIgG-Fc-C) H 3) Selecting an amino acid sequence Ser383-Asn389 (SNGQPEN) in a binding region as a simulation object, constructing a cyclic peptide library with Cys as a connecting residue by utilizing three-dimensional structural information of the amino acid sequence Ser383-Asn389 (SNGQPEN), sequentially carrying out molecular docking (two times of semi-flexible molecular docking) on polypeptide molecules in the polypeptide library and hIgG-Fc fragments by adopting semi-flexible molecular docking programs LeDock and FlexX, selecting a polypeptide molecule with higher binding free energy according to a scoring score, then carrying out molecular dynamics simulation on the polypeptide sequence obtained by a primary screening and the Fc fragments by utilizing an Amber software program, removing the polypeptide sequence which cannot be stably combined with the Fc fragments, and then carrying out experimental verification part, namely verifying the actual combination condition and purification effect between the polypeptide sequence obtained by simulation and the hIgG by an affinity chromatography test, so as to determine the actual binding condition and purification effectAn affinity cyclic peptide ligand to which hIgG specifically binds.
The specific technical scheme of the invention is the application of the hydrophobic cyclic peptide ligand for purifying human immunoglobulin G, wherein the sequence of the hydrophobic cyclic peptide ligand is- [ HWG-C-AKTE ] -, - [ YF-C-WRHE ] -, - [ WV-C-LHHYF ] -, - [ PYF-C-TIE ] -, and- [ FY-C-DEHL ] -, and the sequence of the cyclic peptide ligand with the best purifying effect is- [ HWG-C-AKTE ] -.
The hydrophobic cyclic peptide ligand is obtained through computer simulation bionic design, and the bionic design method comprises the following steps: 1) Constructing a simulation system: selecting crystal conformation of protein A (SpA) -human IgG (hIgG) complex with PBD database ID of 1FC2, and extracting C on protein A and hIgG-Fc fragment H 3 a binding region, wherein the amino acid sequence of the binding region is Ser383-Asn389 (SNGQPEN), and the three-dimensional coordinates of the binding region are selected as coordinate information of a simulation system; 2) Obtaining a bionic polypeptide library: calculating the distance between residues in Ser383-Asn389 sequence by NAMD software, inserting 5-7 amino acid residues X around Cys with Cys as connecting residue to form cyclic polypeptide chain- [ XXX-C-XXXXXX ]]-、-[XX-C-XXXX]-、-[XX-C-XXXXX]-、-[XXX-C-XXX]-、-[XX-C-XXX]Amino acid positioning is carried out by adopting an AutoDock molecular docking program, the type of amino acid residue X is screened and determined, X represents common amino acid residues in 19 except Cys, and a GROMOS program is called to obtain a bionic polypeptide library containing cyclic 6 peptide, cyclic 7 peptide and cyclic 8 peptide sequences; 3) Primary screening of polypeptide libraries: semi-flexibly butting the polypeptide sequences in the polypeptide library with the hIgG-Fc fragment by using LeDock molecular docking software; 4) Rescreening of polypeptide libraries: carrying out semi-flexible molecular docking on the polypeptide sequence obtained in the step (3) through preliminary screening and the Fc fragment again by using FlexX software; 5) Molecular dynamics simulation screening: performing molecular dynamics simulation on the polypeptide series obtained in the step (4) by using Amber software package, and eliminating polypeptide sequences which cannot be stably combined with the Fc fragment; 6) Loading the polypeptide sequence obtained in the step (5) into a column, performing affinity chromatography purification hIgG research, calculating the purity and recovery rate of the purified hIgG, and determining the effective affinity peptide ligand capable of performing stable hydrophobic binding with the hIgG.
Preferably, in the step (3), selecting the polypeptide molecules with the binding free energy lower than-6.0 kcal/mol for the next round of screening according to the docking score; and (4) selecting polypeptide molecules with binding free energy lower than-7.0 kcal/mol according to the scoring score for next round of screening.
Step (2) obtaining a peptide library containing 4256 polypeptide library sequences in total, and selecting polypeptide molecules (1135 polypeptide molecules in total) with affinity binding force lower than-6.0 kcal/mol according to the butt joint scoring score in step (3) for next round of screening; in the step (4), polypeptide molecules with affinity binding force lower than-7.0 kcal/mol (157) are selected according to the scoring score to carry out the next round of screening, 10 polypeptide sequences which can have stable running tracks with hIgG-Fc are selected through MD simulation screening, and effective affinity peptide ligands which can be specifically bound with hIgG are finally selected through an affinity chromatography test: the sequences are- [ HWG-C-AKTE ] - (pep 1 for short), - [ FY-C-WRHE ] - (pep 2 for short), - [ WV-C-LHHYF ] - (pep 3 for short), - [ PYF-C-TIE ] - (pep 4 for short), - [ FY-C-DEHL ] - (pep 5 for short).
The simplified structural formula of the above 5 affinity peptide ligands is as follows:
cys residues are selected to facilitate immobilization of the polypeptide sequence onto the chromatographic medium (Thiopropyl Sepharose B), and the solid sphere represents the chromatographic medium and the bond between the solid sphere and Cys represents the disulfide bond within the structural formula of the peptide ligand. After the polypeptide is connected to Thiopropyl Sepharose B medium, the chromatographic medium can be packed into a column for affinity chromatography purification experiment.
The present invention differs from the ligand design method and results adopted by the university of Tianjin Sun Yan professor task group in that:
1) First, the invention selects C on protein A and hIgG-Fc fragment, which are different from the simulation system of the invention H The three-dimensional sequence of Ser383-Asn389 (SNGQPEN) in the 3-binding region, instead of the 6 key residues on SpA (F132, Y133, H137, E143, R146 and K154) as the modeled target, namelyThe three-dimensional structure of the template protein used for butt joint is fundamentally different.
2) The invention adopts different molecular docking software, the LeDock molecular docking software is particularly suitable for docking of a protein ligand system, ions, metals, water molecules and the like in the protein can be removed by using a charmm force field, the condition of the LeDock on docking conformation score is superior to AutoDock Vina, the optimized conformation of the ligand is used as the input of molecular docking, the performance (57.4%) of the LeDock is superior to AutoDock Vina (49.0%) according to the structure of the conformation with the highest score, namely, the LeDock is more suitable for docking of hIgG and a peptide ligand system, the sampling algorithm can better evaluate the performance of the ligand in a database, the sampling algorithm and the scoring function are the most critical calculation modules of the docking software, and the accuracy of a docking result is decisively influenced.
3) The method is characterized in that FlexX adopted by the polypeptide library compound sieve is semi-flexible butting software, the conformation of peptide ligand can be changed within a certain range in the butting process by adopting FlexX, but the conformation change adjustment is limited to a certain extent, such as certain non-key bond length, bond angle and the like, the conformation of hIgG-Fc fragment is fixed, the FlexX butting method is compatible with calculation efficiency and precision, flexX butting method is flexible butting, the conformations of ligand and template protein can be changed in the butting process, the flexible butting can accurately determine the recognition condition among molecules, but the change of the conformations of the template protein and the ligand causes great calculated amount, lower speed and low efficiency, and how to accurately simulate the conformation change of the macromolecular protein is also a common difficulty faced by the butting software at present, and the grand teaching subject group has been disclosed: spA-IgG binding is predominantly dominated by hydrophobic and hydrogen bonding, polypeptides are designed based on SpA-IgG complexes, but the subject group of polypeptides have been found to have a major difference in binding mechanism with respect to IgG due to the large difference in binding mechanism with protein A, and analysis is due to limitations of Flexpectpdock docking software, which has been reported to demonstrate that molecular docking tends to underestimate hydrophobic interactions between polypeptides and target proteins, e.g., pi-pi interactions between aromatic groups, as calculated using Flexpectpdock software, by default to 0. Therefore, based on the dual consideration of docking efficiency and accuracy, the invention adopts FlexX semi-flexible docking software instead of Flexpdock full-flexible docking, flexX adopts an empirical scoring function, and comprehensively considers hydrophobic interaction, electrostatic interaction and hydrogen bond, and pairwise preference and geometric complementarity among residues.
4) The invention carries on the procedure that the Molecular Dynamics (MD) imitates and adopts is AMBER and not GROMOS, MD imitates and can imitate the action mechanism between the molecules on the atomic level, can obtain the multiple properties of the imitative system through MD imitative protein-ligand system's orbit, such as molecular conformation, energy, dynamics property and interaction energy among protein-ligand, etc., the invention adopts AMBER imitative method to be based on its method to process cyclic peptide more simple and accurate than GROMOS, the simulation of cyclic peptide is distinguished from straight-chain peptide, cyclic peptide end to end, need to process while reading AMBER or GROMOS, otherwise will be regarded as N-end and C-end residue to process automatically, AMBER processes the way to treat the end amino acid of the pdb file of the polypeptide as the common amino acid residue in the middle end, make each amino acid in the ring have no particularities, GROMOS processes the way to end amino acid and carboxyl, define the way to process amino and carboxyl in the force field, cyclic peptide residue is apt to be regarded as N-end and C-end residue, need to process additional, the complex precision is limited.
5) The cyclic peptide ligand prepared by the invention has low flexibility compared with a linear peptide molecule, so that the entropy loss is less when the cyclic peptide ligand is combined with a target protein, higher binding affinity can be obtained, the lower flexibility also causes a specific conformation of the lockable target molecule, and the binding specificity is increased compared with the linear peptide.
6) The affinity between the cyclopeptide ligand and hIgG obtained by the invention is mainly hydrophobic interaction, and then electrostatic interaction and hydrogen bonding are performed. The invention is based on the hydrophobic action model between protein A and IgG, correspondingly obtains the affinity peptide ligand taking the hydrophobic action as the dominant, further verifies the effectiveness of molecular simulation design, and grand professor subjects group obtains the linear polypeptide sequence based on the protein A model to be combined with hIgG based on electrostatic action, however, the electrostatic action is not specific adsorption, other biomass molecules such as albumin in serum are easy to adsorb, and the purity of the obtained hIgG is difficult to achieve commercial utilization.
Drawings
FIG. 1 is an experimental plot of affinity chromatography adsorption of pep1 medium to hIgG under the following conditions: buffer solution (20 mmol/L PBS, pH 7.4) is used for adsorption, and 2% acetic acid aqueous solution is used for elution;
FIG. 2 is an experimental plot of affinity chromatography adsorption of pep2 medium to hIgG under the following conditions: buffer solution (20 mmol/L PBS, pH 7.4) is used for adsorption, and 2% acetic acid aqueous solution is used for elution;
FIG. 3 is an experimental plot of affinity chromatography adsorption of pep3 medium to hIgG under the following conditions: buffer solution (20 mmol/L PBS, pH 7.4) is used for adsorption, and 2% acetic acid aqueous solution is used for elution;
FIG. 4 is an experimental plot of affinity chromatography adsorption of pep4 medium to hIgG under the following conditions: buffer solution (20 mmol/L PBS, pH 7.4) is used for adsorption, and 2% acetic acid aqueous solution is used for elution;
FIG. 5 is an experimental plot of affinity chromatography adsorption of pep5 medium to hIgG under the following conditions: buffer solution (20 mmol/L PBS, pH 7.4) is used for adsorption, and 2% acetic acid aqueous solution is used for elution;
FIG. 6 is an experimental plot of affinity chromatography adsorption of pep6 media to hIgG under the following conditions: buffer solution (20 mmol/L PBS, pH 7.4) is used for adsorption, and 2% acetic acid aqueous solution is used for elution;
FIG. 7 is an experimental plot of affinity chromatography adsorption of pep1 medium to hIgG under the following conditions: the adsorption adopts buffer solution (20 mmol/L PBS, pH 7.4), and the elution adopts 0.5mol/LNaCl aqueous solution and 2% acetic acid aqueous solution in sequence;
FIG. 8 is an experimental plot of affinity chromatography purification of hIgG from human serum by pep1 affinity chromatography media;
FIG. 9 is a gel electrophoresis of pep1 affinity chromatography media for purification of hIgG from human serum.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings, the following examples being intended to illustrate, but not to limit the invention in any way.
Example 1 construction of a simulation System
Selecting crystal conformation of SpA-hIgG1 complex with PBD database ID of 1FC2, extracting C on SpA and hIgG-Fc fragment H 3, the amino acid sequence of the binding region is Ser383-Asn389 (SNGQPEN), and the three-dimensional coordinates of each amino acid residue of the binding region are selected as coordinate information of a simulation system.
Example 2 obtaining a bionic polypeptide library
Calculating the distance between each residue in Ser383-Asn389 sequence by NAMD software, taking Cys as a connecting residue, inserting 5-7 amino acid residues X around the Cys to form a cyclic multi-titanium chain- [ XXX-C-XXXX ] -, - [ XX-C-XXXXXX ] -, - [ XX-C-XXX ] - [ XXX-C-XXX ] -, and carrying out amino acid positioning by adopting an AutoDock molecular docking program, determining the type of the amino acid residue X which is matched with the action of the Ser383-Asn389 coordinate system and has the best binding position, wherein X represents 19 common amino acid residues except Cys, calling the GROMOS program to obtain a bionic polypeptide library of cyclic 6 peptide, cyclic 7 peptide and cyclic 8 peptide sequences which can be stably bound with hIgG-Fc and are connected end to end, and the bionic polypeptide library contains 4256 sequences.
EXAMPLE 3 Primary screening of polypeptide libraries
And (3) carrying out molecular docking on polypeptide molecules in the polypeptide library with the hIgG-Fc three-dimensional structure sequentially by using LeDock molecular docking software, wherein docking results show that most of the polypeptides can be combined with Fc fragments, the binding affinity is distributed between-3.5 and-7.6 kcal/mol, the size of the affinity and the screening efficiency are comprehensively considered, and finally, polypeptide molecules (1135 total) with the binding affinity lower than-6.0 kcal/mol (the absolute value of the affinity is larger than 6.0 kcal/mol) are selected for next round of screening.
EXAMPLE 4 rescreening of polypeptide libraries
And (3) respectively carrying out semi-flexible molecular docking on 1135 polypeptide sequences obtained in the step (3) through FlexX software, wherein docking results show that all polypeptides can be combined with Fc fragments, the binding affinity is distributed between-5.8 and-8.5 kcal/mol, and finally, polypeptide molecules (157 total polypeptide molecules) with the binding affinity lower than-7.0 kcal/mol (the absolute value of the affinity is larger than 7.0 kcal/mol) are selected for next round of screening. The binding conformation and affinity between the ligand and the protein are screened again by using FlexX in the butt joint, so that the false positive affinity ligand displayed by the butt joint of the LeDock molecules is eliminated, and the accuracy is further enhanced.
Example 5 molecular dynamics simulation screening
Molecular docking is a simulation of the static binding conformation between polypeptide and protein, and in order to further verify the affinity binding force between pep-hIgG, the dynamic binding behavior between polypeptide and protein is also considered, so that molecular dynamics simulation is required to study the binding dynamic information between polypeptide and protein. Carrying out molecular dynamics simulation on the 157 polypeptide sequences obtained in the step (4) by using Amber software package program, wherein the dynamic process of Amber operation is divided into three steps: energy minimization, system balance and actual kinetic simulation. Wherein the energy minimization is divided into two steps, the first step is mini1: limiting protein, minimizing partial energy of solvent, and in the second step mini2: relaxing proteins, minimizing overall system energy; the system balance is also divided into two steps: the first step, the system is heated under the constraint protein, the system is heated, the dynamics of 20ps are operated from 0K to 300K, the system is boosted in the second step, and the constant temperature and constant pressure dynamics calculation of 100ps is operated for balancing the system; thirdly, actually simulating to obtain a track, and running 10ns molecular dynamics simulation. During the simulation, it was found that part of the polypeptide molecules gradually detached during the contact with the Fc fragment (10 ns), i.e. they could not stably bind to the Fc fragment, and finally 10 polypeptide sequences were selected which had a stable running track with hIgG-Fc: [ HWG-C-AKTE ] - (pep 1), - [ FY-C-WRHE ] - (pep 2), - [ WV-C-LHHYF ] - (pep 3), - [ PYF-C-TIE ] - (pep 4), - [ FY-C-DEHL ] - (pep 5), - [ MEY-C-KAGE ] - (pep 6), - [ VY-C-LEIT ] - (pep 7), - [ FD-C-TPHRA ] - (pep 8), - [ PHR-C-GAV ] - (pep 9), - [ FW-C-STPR ] - (pep 10). The control experiment used pep6 peptide ligand.
Test section
Main raw materials and equipment: polypeptide molecules, jier Biochemical (Shanghai) limited, synthesized by solid phase synthesis; thiopropyl Sepharose 6B Medium, GE healthcare; crosslinking the cyclopeptide molecule to Thiopropyl Sepharose B medium to produce a polypeptide ligand medium (density: 10. Mu. Mol/g); hIgG, sigma Co; human serum, beijing Ding national biotechnology Limited company; AKTA Purifier 10 chromatograph, GE healthcare; tricorn type chromatographic column, HR5/5, GE healthcare.
Example 6
Peptide ligand medium (pep 1-Sepharose, pep 2-Sepharose, pep 3-Sepharose, pep 4-Sepharose, pep 5-Sepharose, pep 6-Sepharose) was loaded onto a chromatographic column, and before loading, an adsorption buffer (20 mmol/L PBS, pH 7.4) was used to equilibrate well, after stabilization of the UV baseline, 100. Mu.L of hIgG (1.0 mg/mL) dissolved in the adsorption buffer was pulsed into the column at a flow rate of 50. Mu.L/min, after protein injection, adsorption buffer was continuously injected into the column, the unadsorbed protein was washed, and the adsorbed protein was eluted with 2% aqueous acetic acid at a flow rate of 0.8 mL/min, and the eluate was examined for absorbance at 280nm using a UV-900 detector. pep1-Sepharose was additionally tested for chromatography with 0.5 mol/LNaCl. The chromatographic test results are shown in FIGS. 1 to 7.
From the chromatograms, only trace protein in the effluent fractions of FIGS. 1-5 is eluted, which shows that hIgG can be fully bound to pep 1-pep 5 affinity medium under the adsorption condition of pH7.4, the bound protein can be washed out by 2% acetic acid aqueous solution, and FIG. 7 shows that NaCl solution can not elute the adsorbed protein, which shows that the binding between peptide ligand and hIgG is mainly hydrophobic and the same binding mechanism as that of protein A-hIgG, which also directly proves the accuracy of the molecular simulation design of affinity peptide ligand of the invention. In FIG. 6, a large amount of protein was directly eluted after sample injection, and only a small amount of protein was contained in the eluted fraction, indicating that pep6 was not able to bind to hIgG efficiently and was not able to act as an affinity peptide ligand for hIgG.
Example 7 static adsorption isotherm assay
Pre-balancing the wet and dry medium (pep-Sepharose 6B medium) with buffer solution, weighing 10mg of the dry medium, mixing with 1.0mL of hIgG solution (adsorption buffer solution, 0.1-5 mg/mL), reacting at 150rpm for 5 hr, centrifuging the adsorption system at 2000rpm for 6min, collectingTaking supernatant, measuring absorbance of the supernatant at 280nm by using a spectrophotometer to determine protein concentration [ (]c) Protein adsorption Density [ ]q) By mass balance calculation, langmuir model is appliedq=q m c/(K d +c) Describing adsorption isotherms in whichq m Is the adsorption capacity (mg-protein/g-wet-on-pump medium),K d is the apparent dissociation constant. The measurement results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the adsorption capacity of pep6 medium for hIgG was very low, only 3.8 mg/g, again demonstrating that it is not an effective affinity peptide ligand for hIgG, which corresponds to the affinity chromatography test results in example 5.
The adsorption capacity of pep1 to pep5 affinity media was 91.1-136.4 mg/g, the adsorption capacity of pep4 (91.1 mg/g) was higher than FYCHWALE and FYCHTIDE, and the adsorption capacity of the remaining affinity peptide ligands for hIgG was higher than that of the optimal peptide ligand FYWHCLDE (100.5 mg/g) obtained from the grandchild professor task group, indicating that the higher the adsorption capacity, the stronger the adsorption capacity of the ligands for proteins.K d The dissociation constant is represented by a value representing,K d smaller indicates higher affinity between peptide ligand and protein, and it can be seen that pep1 and pep2 have the highest affinity, better than fywhcde, and pep3-pep5 have better affinity than FYCHWALE and FYCHTIDE. From the above data, it can be seen that the binding affinity of pep1 to pep5 affinity peptide ligands for hIgG is overall superior to three linear octapeptide ligands of the grandchild professor problem group.
EXAMPLE 8 affinity chromatography purification of hIgG in human serum
According to the experimental results of examples 5-6, the optimal affinity peptide ligand pep1 was determined, human IgG in serum was purified using HR5/5 column loaded with 1mL pep1 affinity chromatography medium, the serum samples were diluted (10-fold diluted) with adsorption buffer (20 mmol/L PBS, pH 7.4) and subjected to membrane filtration to remove insoluble material, the columns were equilibrated well with the adsorption buffer until UV baseline leveled, 500. Mu.L of human serum diluent (total protein concentration 8.86 mg/mL) was injected at a flow rate of 0.5mL/min, the column was then washed with the adsorption buffer, the adsorbed proteins were eluted with 2% aqueous acetic acid, the effluent fractions (FT-1, FT-2) and the elution fractions (E-1, E-2) were collected, SDS-Gel electrophoresis was performed, the protein recovery was detected with Micro-BCA kit, and protein purity was analyzed using Gel-Pro Analyzer software. The test results are shown in fig. 8 and 9.
As can be seen from the electrophoretogram, the effluent fraction contained a large amount of human serum albumin, only a trace amount of human IgG and a small amount of other impurities, hIgG was completely eluted with aqueous acetic acid (2%), the eluted fractions E-1 and E-2 were mixed, and the purity (96%) and recovery rate (92%) of hIgG were calculated to be higher than those of purified hIgG from human serum by FYWHCLDE affinity medium (90%) and recovery rate (87%).
In summary, the experimental characterization results of the invention show that the cyclic peptide ligand designed by the invention has purification effect on human IgG in terms of adsorption capacity, binding affinity, binding purification and recovery rate, and the like, and is better than FYWHCLDE, FYCHWALE, FYCHTIDE peptide ligand medium on the whole.
That is, in comparison with the rational design method proposed by the grand professor task group, the molecular simulation design of the present invention uses a simulation system, molecular docking software, molecular dynamics simulation program, and simulation parameters that are different from those of the task group, and the shape of the peptide ligand to be screened, the effect with hIgG (apparent buffer used for adsorption and elution), and the binding force and affinity for hIgG are different. Therefore, the two are not comparable, and the purification effect of the cyclic peptide ligand of the present invention is combined, and the overall effect is superior to that of the ligand obtained by the subject group.
Claims (3)
1. Use of a hydrophobic cyclic peptide ligand for purifying human immunoglobulin G, characterized in that: the sequence of the hydrophobic cyclic peptide ligand is- [ HWG-C-AKTE ] -, - [ YF-C-WRHE ] -, - [ WV-C-LHHYF ] -, - [ PYF-C-TIE ] -, and- [ FY-C-DEHL ] -;
the hydrophobic cyclic peptide ligand is obtained by a computer simulation bionic design method, and the bionic design method comprises the following steps:
(1) Constructing a simulation system: selecting crystal conformation of protein A (SpA) -human IgG (hIgG) complex with PBD database ID of 1FC2, and extracting C on protein A and human IgG-Fc fragment H 3 a binding region, wherein the amino acid sequence of the binding region is Ser383-Asn389 (SNGQPEN), and the three-dimensional coordinates of the binding region are selected as coordinate information of a simulation system;
(2) Obtaining a bionic polypeptide library: calculating the distance between residues in Ser383-Asn389 sequence by NAMD software, taking Cys as a connecting residue, inserting 5-7 amino acid residues X around the Cys to form a cyclic polypeptide chain- [ XXX-C-XXXX ] -, - [ XX-C-XXXXXX ] -, - [ XX-C-XXXXX ] - [ XXX-C-XXX ] -, and carrying out amino acid positioning by adopting an Autodock molecular docking program, screening to determine the type of the amino acid residues X, wherein X represents the common amino acid residues in 19 except Cys, and calling a GROMOS program to obtain a bionic polypeptide library containing cyclic 6 peptide, cyclic 7 peptide and cyclic 8 peptide sequences;
(3) Primary screening of polypeptide libraries: semi-flexibly butting polypeptide sequences in a polypeptide library with a human IgG-Fc fragment by using LeDock molecular docking software;
(4) Rescreening of polypeptide libraries: carrying out semi-flexible molecular docking on the polypeptide sequence obtained in the step (3) through preliminary screening and the Fc fragment again by using FlexX software;
(5) Molecular dynamics simulation screening: performing molecular dynamics simulation on the polypeptide series obtained in the step (4) by using Amber software package, and eliminating polypeptide sequences which cannot be stably combined with the Fc fragment;
(6) Loading the polypeptide sequence obtained in the step (5) into a column, researching the purification of human IgG by affinity chromatography, calculating the purity and recovery rate of the purified human IgG, and determining the effective affinity peptide ligand capable of carrying out stable hydrophobic binding with the human IgG.
2. The use of a hydrophobic cyclic peptide ligand as defined in claim 1 for purifying human immunoglobulin G, wherein in step (3) the polypeptide molecules having a binding free energy below-6.0 kcal/mol are selected for the next round of screening based on the docking score.
3. The use of a hydrophobic cyclic peptide ligand as defined in claim 1 for purifying human immunoglobulin G, wherein in step (4) the polypeptide molecules having a binding free energy below-7.0 kcal/mol are selected for the next round of screening according to the score.
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