CN116254283A - Preparation method of transmembrane domain of single transmembrane protein - Google Patents

Abstract

The invention provides a preparation method of a transmembrane domain of a single transmembrane protein. Aiming at the problem that the single transmembrane protein is very difficult to express and purify by heterologous recombination in the transmembrane domain in the prior art, the inventor comprehensively considers various factors and demonstrates by repeated experiments, establishes a novel recombination expression, purification and cleavage processing system by utilizing TrpLE and GSHHW fused with the transmembrane domain of the single transmembrane protein, ensures that the transmembrane domain of the single transmembrane protein can be well expressed, purified and post-processed, and has no toxicity, no harm and no side reaction.

Description

Preparation method of transmembrane domain of single transmembrane protein

Technical Field

The invention belongs to the field of protein expression and purification, and in particular relates to a preparation method of a transmembrane domain of a single transmembrane protein.

Background

Single-transmembrane proteins (SPTMPs) are important membrane proteins in organisms, and are involved directly or indirectly in regulating a variety of cellular activities such as cellular metabolism, adhesion, migration, growth and death. Single transmembrane proteins are generally divided into three parts, the extracellular domain (extracellular domain, ECD), the transmembrane domain (transmembrane domain, TMD) and the intracellular domain (intracellular cytoplasmic domain, ICD). For a long time, it has been widely accepted that the transmembrane domain of a single transmembrane protein acts only as an anchor for the membrane and has little effect on protein function. However, some recent researches break the previous cognition, and prove that the transmembrane domain of the single transmembrane protein plays an important role in regulating and controlling the protein in the process of functioning. For example, during the functioning of the cell epidermal growth factor (epidermal growth factor receptor, EGFR), the amino terminus of its transmembrane region modulates this receptor activity, and if this region is mutated, EGFR activation will be inhibited. For another example, after death receptor 5 (DR 5) binds to its ligand tumor necrosis factor apoptosis-inducing ligand (TRAIL), apoptosis is induced, and DR 5-mediated signaling is controlled by oligomerization of its transmembrane region, and activation of DR5 is inhibited if the critical amino acid site at the oligomerization interface of the transmembrane region is disrupted. In addition, there have been studies showing that transmembrane domain function is associated with substrate binding or disease development. These studies demonstrate from a number of aspects that the transmembrane region plays an important role in the regulation of transmembrane protein activity and also show the potential for transmembrane proteins as drug targets.

However, due to the unique biochemical nature of the transmembrane domain of single-pass transmembrane proteins, current research into the mechanism of transmembrane domain regulated molecules has been severely retarded. The short length of the transmembrane domain (i.e. transmembrane helix) of the single transmembrane protein (a polypeptide chain consisting of only about 20 amino acids) and the high hydrophobicity make heterologous recombinant expression and protein purification difficult, and bring about great challenges to structural biology research. The structural biology studies of transmembrane helices have been published in a number of papers, mainly by nuclear magnetic resonance (nuclear magnetic resonance, NMR), by means of the escherichia coli expression system, with a Methionine residue (M) as a spacer, fusing the transmembrane helix with the carboxy-terminus of the TrpLE tag protein (i.e. TrpLE-M-transmembrane helix). TrpLE is a pro-expression tag protein which has been shown to be very efficient in helping transmembrane helix expression in E.coli inclusion bodies. A large number of fusion proteins expressed in inclusion bodies were first purified by metal chelate chromatography, followed by cleavage of the fusion protein using cyanogen bromide (CNBr). CNBr can specifically cleave peptide bond at methionine alpha-carboxyl side in protein, and TrpLE-M-transmembrane helix fusion protein will cleave from methionine residue after cleavage by CNBr. The transmembrane helix can be separated from the TrpLE tag by reverse-phase high-performance liquid chromatography (reverse-phase high-performance liquid chromatography) to obtain high-purity polypeptide sample.

The TrpLE fusion transmembrane helix protein purification method greatly promotes the biological research of the transmembrane domain structure of single transmembrane protein, and more transmembrane helices are purified and analyzed by the method. However, the method of cutting the TrpLE-M-transmembrane helix using CNBr still has some significant disadvantages: (1) CNBr is a highly toxic substance, has strong inhalation and contact toxicity, an operator needs to operate in a fume hood and protect the fume hood, CNBr is harmful to the environment, waste liquid generated in the cutting and dialysis processes needs to be carefully treated, corresponding special treatment is needed, and the whole step is complicated; (2) Since CNBr specifically recognizes and cleaves the alpha-carboxy terminal peptide bond of methionine, all methionine present in the transmembrane helix needs to be mutated in order to prevent additional cleavage reactions; (3) Serine and threonine should be avoided in the vicinity of the cleavage site methionine, since the hydroxyl groups of the two amino acid side chains undergo side reactions with CNBr to produce stable intermediates that interfere with the cleavage reaction; (4) The reaction for CNBr cleavage is usually carried out in 70% formic acid solution, a condition which is too extreme for proteins and may cause side reactions.

Based on the defects of the traditional CNBr-cutting TrpLE-M-transmembrane helix method, a new non-toxic and harmless method with smaller side reaction is needed to be developed.

Disclosure of Invention

The invention aims to provide a preparation method of a transmembrane domain of a single transmembrane protein.

In a first aspect of the present invention, there is provided a method for preparing a transmembrane domain of a single transmembrane protein, comprising: (1) Creating an expression construct that expresses a fusion protein comprising the following elements in operative linkage: a transmembrane domain that facilitates expression of the tag-GSHHW pentapeptide-single transmembrane protein; (2) Introducing the expression construct of (1) into a prokaryotic host cell for expression to obtain an expression and purification product; (3) Cutting the transmembrane domain of the expression promoting label-GSHHW pentapeptide-single transmembrane protein, and separating to obtain the transmembrane domain of the single transmembrane protein; wherein the system for cutting contains guanidine hydrochloride, tris, sodium chloride, acetone oxime, dodecyl phosphatidylcholine and nickel chloride.

In one or more embodiments, the pro-expression tag is a tag that facilitates expression of the transmembrane domain of a single transmembrane protein in a prokaryotic host cell; preferably, the expression-promoting tag is TrpLE.

In one or more embodiments, the prokaryotic host cell is an E.coli cell.

In one or more embodiments, in step (2), the protein is expressed to form inclusion bodies, followed by denaturation.

In one or more embodiments, in step (3), the system for cutting comprises:

guanidine hydrochloride: 2+ -1M; tris: 25.+ -.10 mM;

sodium chloride: 150.+ -.50 mM; acetone oxime: 100.+ -.30 mM;

dodecyl phosphatidylcholine: 1+/-0.5%; nickel chloride: 2.+ -. 1mM.

In one or more embodiments, the system for cutting comprises:

guanidine hydrochloride: 2+ -0.5M; tris: 25+ -5 mM;

sodium chloride: 150.+ -.25 mM; acetone oxime: 100+ -15 mM;

dodecyl phosphatidylcholine: 1+/-0.3 percent; nickel chloride: 2.+ -. 0.5mM.

In one or more embodiments, the system for cutting comprises:

guanidine hydrochloride: 2+ -0.3M; tris: 25+ -3 mM;

sodium chloride: 150.+ -.15 mM; acetone oxime: 100+ -10 mM;

dodecyl phosphatidylcholine: 1+/-0.2 percent; nickel chloride: 2.+ -. 0.3mM.

In one or more embodiments, the system for cutting comprises:

guanidine hydrochloride: 2+ -0.2M; tris: 25+ -2 mM;

sodium chloride: 150.+ -.10 mM; acetone oxime: 100+ -5 mM;

dodecyl phosphatidylcholine: 1+/-0.1 percent; nickel chloride: 2.+ -. 0.2mM.

In one or more embodiments, the expression construct is an expression vector; preferably prokaryotic expression vectors.

In one or more embodiments, in step (3), the cutting is performed: at a temperature of 37.+ -. 5 ℃, preferably 37.+ -. 3 ℃, more preferably 37.+ -. 2 ℃, more preferably 37.+ -. 1 ℃.

In one or more embodiments, in step (3), the cutting is performed: at a pH of 7.5 to 9.2, preferably 7.6 to 9.0, more preferably 7.7 to 8.5, more preferably 7.8 to 8.3, more preferably 7.9 to 8.2.

In one or more embodiments, the transmembrane domain of the single transmembrane protein comprises: the transmembrane segment of the Spike protein (Spike-TMD), the transmembrane domain of the T cell surface receptor beta (TCR beta-TMD).

In another aspect of the invention, there is provided a recombinant expression vector comprising an expression construct comprising the following elements operably linked: the expression promoting tag coding gene-GSHHW pentapeptide coding gene-single transmembrane protein transmembrane domain coding gene; preferably, the expression-promoting tag is TrpLE.

In one or more embodiments, the coding gene is a codon optimized coding gene according to the expression host.

In another aspect of the invention, a prokaryotic host cell is provided, comprising an expression construct comprising the following elements operably linked: the expression promoting tag coding gene-GSHHW pentapeptide coding gene-single transmembrane protein transmembrane domain coding gene; preferably, the prokaryotic host cell is an E.coli cell.

In another aspect of the invention, there is provided the use of said recombinant expression vector or said prokaryotic host cell for recombinant expression of the transmembrane domain of a single transmembrane protein.

In another aspect of the invention, there is provided a kit for recombinant expression of a transmembrane domain of a single transmembrane protein comprising: the recombinant expression vector; a prokaryotic host cell; and a cleavage system comprising guanidine hydrochloride, tris, sodium chloride, acetoxime, dodecyl phosphatidylcholine and nickel chloride.

In another aspect of the invention, there is provided a kit for recombinant expression of a transmembrane domain of a single transmembrane protein comprising: the prokaryotic host cell; and a cleavage system comprising guanidine hydrochloride, tris, sodium chloride, acetoxime, dodecyl phosphatidylcholine and nickel chloride.

In one or more embodiments, the kit further includes (but is not limited to): IPTG, inclusion body denaturation (e.g., guanidine hydrochloride buffer), triton X-100, nacl, ni column and packing therefor, imidazole, elution buffer and/or culture medium (etc.).

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, schematic representation of cleavage of TrpLE-pentapeptide fusion proteins by nickel ions;

(a) Schematic sequence diagram of TrpLE-pentapeptide fusion protein through nickel ion cleavage;

(b) Plasmid schematic diagram of TrpLE-pentapeptide fusion protein expression vector and gene and protein sequences corresponding to key sites;

(c) The TrpLE-pentapeptide fusion protein is compared with the escherichia coli lysate before and after induction by using 0.5mM IPTG, and the arrow indicates the expression of the TrpLE fusion protein;

(d) The elution protein (mainly TrpLE fusion protein) obtained after the TrpLE fusion protein passes through metal ion chelate chromatography, and the arrow shows the band of the TrpLE fusion protein.

FIG. 2, vs. Ni ²⁺ Optimizing a method for cutting a pentapeptide sequence;

(a) Cleavage results of the TrpLE-pentapeptide fusion protein in different cleavage solutions;

(b) Cleavage results of TrpLE-pentapeptide fusion protein with addition of different detergents (1%DPC, 1.5%Triton X-100,0.5% LMPG,1.6% DHPC) in 2M guanidine hydrochloride solution;

(c) Cleavage results of the TrpLE-pentapeptide fusion protein under different pH conditions;

(d) Cleavage results of the TrpLE-pentapeptide fusion protein under different temperature conditions;

(e) Results of cleavage of the TrpLE-pentapeptide fusion protein at different times;

and (3) cutting: a sample of pre-cleavage TrpLE-pentapeptide fusion protein;

m: protein molecular standard.

FIG. 3 through Ni ²⁺ A flow of cutting the pentapeptide sequence and purifying to obtain a nuclear magnetic resonance experimental sample;

the TrpLE-pentapeptide fusion protein is obtained by the induction expression of an escherichia coli expression system, and then the fusion protein is purified by metal chelate chromatography (Ni-NTA); purified fusion protein in Ni ²⁺ And cutting, namely breaking from the pentapeptide sequence, and returning to the chromatographic column again to obtain a transmembrane helix (TMD) sample for nuclear magnetic resonance experiments.

FIG. 4, spike-TMD and TCR. Beta. -TMD sequence schematic;

the schematic representation of the sequences of the novel coronavirus Spike protein Spike (a) and T cell surface receptor β (b) shows the positions and sequences of the selected transmembrane domains in Spike and TCR proteins in this example. Wherein methionine in the sequence is highlighted in red.

FIG. 5, SEC, SDS-PAGE and TROSY results for Spike-TMD samples;

(a) The SEC result shows that the sample state of Spike-TMD is uniform, and the SDS-PAGE result shows that the purity of the sample is high, so that the purity and uniformity of the sample obtained by the method of the invention are proved to meet the requirement of further structural biological research;

(b) a sample in aProceeding with ¹ H- ¹⁵ Results of the ntrosy experiment.

FIG. 6, MALDI-TOF, SDS-PAGE, TROSY results of TCR β -TMD samples;

(a) MALDI-TOF mass spectrum results show that the molecular weight of the sample of TCR beta-TMD is correct, and SDS-PAGE results show that the purity of the sample is high;

(b) Sample in a ¹ H- ¹⁵ Results of the ntrosy experiment.

Detailed Description

Aiming at the problem that the single transmembrane protein is very difficult to express and purify by heterologous recombination in the transmembrane domain in the prior art, the inventor comprehensively considers various factors and demonstrates by repeated experiments, establishes a novel recombination expression, purification and cleavage processing system by utilizing TrpLE and GSHHW fused with the transmembrane domain of the single transmembrane protein, ensures that the transmembrane domain of the single transmembrane protein can be well expressed, purified and post-processed, and has no toxicity, no harm and no side reaction.

Terminology

As used herein, the terms "pentapeptide" and "pentapeptide tag" are used interchangeably and refer to peptides of the "GSHHW" sequence.

As used herein, the terms "transmembrane domain," "transmembrane helix protein," and "transmembrane domain of a single transmembrane protein" are used interchangeably to refer to a particular membrane protein, without the presence of a repeating transmembrane domain structure. It is derived from a single transmembrane protein, which is generally divided into three parts, the extracellular domain (extracellular domain, ECD), the transmembrane domain (transmembrane domain, TMD) and the intracellular domain (intracellular cytoplasmic domain, ICD).

As used herein, the term "protein of interest" refers to a protein of interest that requires recombinant expression using a host cell. Unless otherwise indicated, the proteins of interest described herein are the transmembrane domains of a single transmembrane protein.

As used herein, the term "expression cassette" or "gene expression cassette" refers to a gene expression system comprising all the necessary elements required for expression of a polypeptide of interest (in the present invention, the transmembrane domain of a single transmembrane protein), typically comprising the following elements: a promoter, a gene sequence encoding a polypeptide, a terminator; optionally signal peptide coding sequences and the like; these elements are operatively connected.

As used herein, the term "construct" or "expression construct" refers to a recombinant DNA molecule that comprises the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is typically contained in an expression vector; this DNA molecule also comprises the appropriate regulatory elements necessary or contemplated for transcription of the operably linked coding sequence in vitro or in vivo. "regulatory element" as used herein refers to a nucleotide sequence that controls the expression of a nucleic acid sequence to some extent.

As used herein, the term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "exogenous" or "heterologous" refers to a relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is "exogenous" to a gene of interest if the combination of the promoter and the sequence of the gene is not normally naturally occurring. The particular sequence is "exogenous" to the cell or organism into which it is inserted. In the present invention, the transmembrane domain of the single transmembrane protein is "exogenous" or "heterologous" with respect to the host.

Expression, purification and cleavage of the transmembrane domain of a single transmembrane protein

Recombinant expression of heterologous proteins is an important research topic in the biotechnology field. In order to increase the expression rate of recombinant proteins, a great deal of laboratory work is often required, and repeated experiments, analysis, summarization and retests are performed, and many factors can affect the expression and purification of recombinant proteins. This is especially true of the transmembrane domain of single-pass transmembrane proteins, which are proteins of relatively short length (polypeptide chains of only about 20 amino acids) and relatively high hydrophobicity and high dynamic properties, and which are difficult to express and purify by heterologous recombination. This presents a great challenge for structural biology research.

The invention discloses an optimized technical scheme, and develops a novel method for purifying and cutting TrpLE fusion transmembrane polypeptide through nickel ion reaction, and provides a novel nontoxic and harmless novel method without side reaction for expression and purification of a transmembrane segment of a membrane protein.

The invention provides a preparation method of a transmembrane domain of a single transmembrane protein, which comprises the following steps: (1) Creating an expression construct that expresses a fusion protein comprising the following elements in operative linkage: a transmembrane domain that facilitates expression of the tag-GSHHW pentapeptide-single transmembrane protein; (2) Introducing the expression construct of (1) into a prokaryotic host cell for expression to obtain an expression and purification product; (3) Cutting the transmembrane domain of the expression promoting label-GSHHW pentapeptide-single transmembrane protein, and separating to obtain the transmembrane domain of the single transmembrane protein; wherein the system for cutting contains guanidine hydrochloride, tris, sodium chloride, acetone oxime, dodecyl phosphatidylcholine and nickel chloride.

In the method, the original TrpLE-M-transmembrane helix expression vector is changed into a sequence glycine-serine-histidine-tryptophan (GSHHHW, pentapeptide) with five amino acid residues by means of molecular cloning. Nickel ion (Ni) ²⁺ ) The protein can be cleaved between glycine and serine. The novel cleavage sequence and the corresponding cleavage method avoid the use of CNBr and simultaneously avoid methionine and adjacent serine threonine mutations which have to be carried out as a result of cleavage of methionine. In the cleavage process, the inventors optimized the purification steps, simplifying the purification method of the transmembrane helix. And purifying the fusion protein expressed in the escherichia coli inclusion body by a metal chelating chromatography method, cutting, then hanging a chromatographic column, and collecting a penetrating fluid to obtain a sample for solution nuclear magnetic resonance research.

In the cleavage conditions described above, tris is the buffer salt required to maintain the pH of the solution, and NaCl provides the normal ionic strength of the salt in solution. Acetoxime is an accelerator of the nickel ion cleavage reaction. Guanidine hydrochloride is a substance required to aid in the dissolution and cleavage of fusion proteins. DPC is a detergent that increases solubility to the transmembrane helix during cleavage and aids in protein folding and renaturation. In biological studies of membrane proteins, detergents are an indispensable class of compounds. The detergent molecule has a hydrophilic head group and a hydrophobic tail long chain. In aqueous solution, the detergent molecules will spontaneously form a globular structure with the head portion held together towards the tail portion, known as micelles (micellar). In the study of membrane proteins, detergents are often added to coat the hydrophobic regions of the membrane proteins with spherical micelles, helping to stabilize the membrane proteins in aqueous solution. DPC is a detergent commonly used in solution nmr studies.

In the specific embodiment of the invention, the inventor constructs the transmembrane polypeptide which needs to be expressed and purified on an expression vector containing a TrpLE-pentapeptide label, expresses inclusion body protein through an escherichia coli expression system, purifies the inclusion body protein by a nickel ion affinity chromatographic column to obtain protein, and obtains the transmembrane polypeptide with high purity after cleavage by a nickel ion reaction.

After obtaining optimized cleavage conditions, the inventors further tested the feasibility of cleaving the TrpLE-pentapeptide fusion protein in DPC and purifying the protein of interest. The fusion protein after cleavage is divided into a TrpLE fragment with a metal chelate chromatography tag and a transmembrane helix (transmembrane domian, TMD) fragment to be studied, and the fusion protein without the metal chelate chromatography tag being cleaved also exists in the whole reaction solution. To further separate the TMD fragment from the cleavage solution system, the mixed sample needs to be subjected to metal chelate chromatography again. The TMD fragment without the metal chelating chromatographic label can flow out rapidly in the chromatographic process, and a TMD sample can be obtained after collecting the flow-through liquid. At this time, the TMD sample is in DPC micelle environment, and after the sample reaches 0.5mM concentration after concentration, the sample can meet the requirement of solution nuclear magnetic resonance (solution NMR) on the sample, and can be directly used for the solution nuclear magnetic resonance experiment. If it is desired to further obtain a protein sample of higher purity and more uniform state, or to modify the micellar environment in which the TMD is located, the protein sample may be further purified by size exclusion chromatography (size exclusion chromatography, SEC).

The inventor has found unexpectedly that adding a proper amount of guanidine hydrochloride into a cutting system has a very remarkable promoting effect on cutting through exploring different cutting environments in the optimization process, and if guanidine hydrochloride is not added, the cutting efficiency of TrpLE is extremely low and effective separation cannot be realized. Guanidine hydrochloride is thus used as an indispensable additive in the cleavage system of the method according to the invention.

Expression construct, host cell and kit

Based on the novel findings of the present invention, the present invention also provides a fusion protein comprising the following elements operatively linked: the expression promoting tag-GSHHW pentapeptide-transmembrane domain of single transmembrane protein.

The invention also provides an expression construct for expressing the fusion protein, and a host cell containing the expression construct. As a preferred mode of the invention, the expression construct is an expression vector.

Methods for introducing exogenous coding genes or expression constructs into cells of the microorganism are well known to those skilled in the art. In general, the coding gene or expression construct is obtained and incorporated into a suitable expression vector and transferred into a microbial cell.

The invention also provides a kit for facilitating laboratory-scale or production-scale expression.

As a preferred mode, the kit comprises: the recombinant expression vector provided by the invention; a prokaryotic host cell; and a cleavage system comprising guanidine hydrochloride, tris, sodium chloride, acetoxime, dodecyl phosphatidylcholine and nickel chloride.

As another preferred mode, the kit includes: the prokaryotic host cell (already carrying the recombinant expression vector) of the invention; and a cleavage system comprising guanidine hydrochloride, tris, sodium chloride, acetoxime, dodecyl phosphatidylcholine and nickel chloride.

Advantageous effects

The transmembrane domain of the single transmembrane protein plays an important role in the aspects of signal transduction, cellular immunity and the like, and is one of main target proteins in the research and development of medicines at present. The transmembrane domain of a single transmembrane protein is typically composed of a transmembrane helix, typically comprising only about 20 amino acid residues, and is highly hydrophobic. More and more researches prove that the transmembrane domain of the single transmembrane protein has a regulatory effect on the function of the protein, and directly or indirectly influences the function mediated by the transmembrane protein, so that the transmembrane domain of the single transmembrane protein becomes a new potential drug target. However, the structural biology of the transmembrane domain of single-transmembrane proteins has been studied with a lag in status, as determined by their unique biochemical properties, compared to other types of proteins. The in vitro recombinant expression difficulty is higher due to the characteristics of smaller molecular weight and high hydrophobicity.

In the current research field, cleavage and purification of the transmembrane helix are usually achieved by cleavage of the methionine residue intermediate the TrpLE and the transmembrane helix in the TrpLE fusion protein with CNBr. However, the limitation of this method is obvious due to the toxicity of CNBr and the requirement for special sites. The invention realizes high-yield expression of TrpLE fusion transmembrane helix by introducing a novel cleavage method pentapeptide and simultaneously passes Ni ²⁺ Cutting and simplifying the subsequent purification steps, and finishing the improvement of the expression method of the transmembrane helix. First, since only millimole grade Ni is required to be added during cutting ²⁺ The toxicity is obviously smaller than that of cutting by CNBr; meanwhile, the cleavage system is in the environment of guanidine hydrochloride solution, but not 70% formic acid, so that side reactions caused by high-concentration formic acid can not occur. Furthermore, since the protein is cleaved through the methionine site, it is required that no methionine is present in the protein sequence except for the cleavage site to prevent additional cleavage. If an endogenous methionine site is present in the sequence under investigation, it is necessary to mutate it. At the same time, the vicinity of methionine cleavage site affects the cleavage efficiency for preventing side reactionsSerine and threonine should also be avoided. The invention solves the embarrassing reality that extra mutation has to be introduced due to cutting. The pentapeptide sequence consists of five amino acids-GSHHW-, and the probability of the sequence to be completely present in more than 20 amino acids in the transmembrane domain is low, so that no additional mutation is needed to prevent the cleavage reaction from occurring outside the cleavage site. Finally, as the cleavage solution contains detergent components, the transmembrane helix can stably exist in the cleavage solution after being cleaved and separated, so that the cleaved sample can be rapidly used for structural biological research after being subjected to simple chromatography again, and the purification step of the transmembrane helix is greatly simplified.

The invention provides a more harmless, simple and easy-to-operate method guidance for in-vitro purification of the transmembrane helix. The method can obtain the transmembrane helix sample more simply so as to meet the requirements of biochemical and structural biological research.

The experimental materials required in the present invention, and the specific experimental procedures for the operation will be described in detail. And further illustrates the practical application of the invention by way of example. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the specific conditions, and are conducted according to conventional experimental procedures (e.g., molecular cloning Experimental guidelines, etc.) or according to the conditions recommended by the materials manufacturer.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Materials and methods

1. Plasmid construction

The original TrpLE-M-transmembrane helix expression vector (original vector is from Blacklow laboratory of university of Harvard, and the basic skeleton of the vector is pMM-LR 6) is changed into a coding sequence consisting of five amino acid residues (pentapeptides) by means of molecular cloning: glycine-serine-histidine-tryptophan (GSHHHW (SEQ ID NO: 1)).

The 1193-1234 (TMD protein) of human Spike-TMD (Uniprot code: P0DTC 2) sequence and the 131-178 (TMD protein) of human TCR beta-TMD (Uniprot code: A0A5B 9) sequence are respectively selected, and after the expression codon optimization of colibacillus is carried out, the target gene is inserted into the HindIII and BamHI restriction enzyme cutting site (plasmid sequence 2236-2248) of the expression vector pMM-LR6 with His-TrpLE-pentapeptide label after modification. The plasmid after correct sequencing was transformed into E.coli DH 5. Alpha. Strain, smeared on LB-agar plates with kanamycin (50. Mu.g/mL) resistance, cultured overnight at 37 ℃, the monoclonal colony was picked up to 5mL LB for 16h, the plasmid was extracted and the concentration was determined, and stored at-80 ℃.

TrpLE sequence is (SEQ ID NO: 2):

KAIFVLKGSLDRDLDSRIELELRTDHKELSEHLLLVDLARNDLARIATP GSRYVADLTKVDRYSYVLHLVSRVVGELRHDLDALHAYRAALNLGTLSGA PKVRA

Spike-TMD sequence (SEQ ID NO: 3):

LNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIML

TCRβ -TMD sequence (SEQ ID NO: 4):

GFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

2. protein expression

0.5 μl (about 80 ng) of the plasmid with correct sequencing was added to E.coli BL21 (DE 3) competent cells, and the mixture was ice-bathed for 30min; after heat shock for 90s at 42 ℃, adding 800 mu L of LB without resistance into an ultra-clean bench after taking out, shaking at 37 ℃ and 200rpm, and activating for 40min; 200. Mu.L of the activated bacteria were plated on kanamycin-resistant plates and incubated overnight in an incubator at 37 ℃. Several single clones were picked from the culture plate and inoculated in 5mL LB medium with kanamycin (50. Mu.g/mL) resistance for small-scale amplification for 12h, when the bacterial liquid was turbid, the bacterial liquid was transferred to 50mL M9 medium at a ratio of 0.1% -0.5%, cultured overnight at 37℃and inoculated to 1L M9 medium. Protein expression was induced by adding Isopropyl Thiogalactoside (IPTG) to a final concentration of 0.5mM at 37℃and 220rpm until the OD 600 was about 0.8. The protein expression temperatures were 25℃respectively. The cells were cultured overnight (18-20. 20 h), and the cells were collected by centrifugation at 4000rpm for 30 min. The cells were resuspended in lysis buffer (50mM Tris,100mM NaCl,pH8.0,1mM PMSF, 50mL buffer per 1L of cells collected in medium) and stirred until the cells were well mixed.

Purification of TrpLE-pentapeptide tag fusion proteins

PMSF (final concentration is 1 mM) is added into the re-suspended bacteria to prevent protein degradation, the bacteria are crushed by a high-pressure homogenizer, the pressure is controlled at 800bar, the bacteria are crushed for 2 times, the bacteria are thoroughly cracked, the obvious reduction of the viscosity of the bacteria liquid can be observed, the low temperature of 4 ℃ is ensured in the process of crushing the bacteria, and the cracked products of the bacteria are placed on ice. The cell lysate was centrifuged at 18000rpm at 4℃for 30min to collect inclusion bodies.

The collected inclusion bodies are dissolved in inclusion body denaturation liquid (40 mL buffer solution is used for the inclusion bodies obtained from the thalli in each 1L culture solution of 6M Guanidine-HCl, 50-mM Tris,100mM NaCl,pH8.0,1%Triton X-100), and in order to improve the dissolution effect, the obtained inclusion bodies can be uniformly grinded in advance by a glass homogenizer and then dissolved overnight. The overnight lysate was centrifuged at 18000rpm at 4℃for 30min and the supernatant was collected. 5mL of pre-equilibrated Ni-NTA beads (equilibration buffer: 6M Guanidine-HCl,50mM Tris,100mM NaCl,pH8.0, 25mM imidozole) was added per liter of bacterial harvest. The balanced Ni-NTA filler was added to the centrifuged supernatant, and imidazole was added at a final concentration of 25mM to inhibit non-specific protein binding. Incubate 2h with stirring at room temperature to allow the fusion protein to bind well to the filler. Pouring the incubated supernatant into an empty chromatographic column, and allowing the supernatant to flow through twice under the action of gravity. Adding 5 times column volume of a hetero-protein eluting buffer (6M Guanidine-HCl,50mM Tris,100mM NaCl,pH8.0, 50mM imidozole) to wash out the hetero-protein with weaker binding; the fusion protein was eluted by adding 5 column volumes of elution buffer (6M Guanidine-HCl,50mM Tris,100mM NaCl,pH8.0, 500mM imidozole). Collecting the fusion protein, adding into 10kDa dialysis bag, and standing in 5L ddH ₂ In O, stirring and dialyzing at 4 ℃, removing the denaturant guanidine hydrochloride solution and imidazole, and rapidly precipitating a large amount of fusion protein. After 2-3h, changeddH ₂ O dialysis, changing water 4 times during dialysis; the fusion protein was obtained for subsequent cleavage experiments by centrifugation at 4000rpm at 4℃for 30 min.

4. Fusion protein cleavage of pentapeptide tags

The fusion protein was resuspended in cleavage buffer (25mM Tris,2M Guanidine-HCl,150mM NaCl,100mM acetone oxime,1%DPC) and vortexed repeatedly to allow sufficient fusion protein to dissolve. Nickel chloride was added at a final concentration of 2mM and the cleavage reaction was carried out at 37℃for 24 hours.

And adding Ni-NTA filler into the cut mixture for metal chelating chromatography to remove TrpLE tag and uncleaved protein, thereby obtaining purified target protein.

Example 1 Spike-TMD expression and cleavage Condition optimization

1. Expression inclusion bodies, denaturation and purification

For the defects of the traditional CNBr-cutting TrpLE-M-transmembrane helix protein method, the inventor conducts a great deal of research, analysis and experimental work to find a method for changing the current situation. After various attempts, the inventors found that prokaryotic expression of transmembrane helicins can be efficiently achieved using "GSHHW" pentapeptides in combination with subsequent optimization of cleavage conditions.

As described in the previous materials and methods, the original coding sequence of methionine at the cutting site in the expression vector of TrpLE-M-transmembrane helix is changed into the coding sequence of GSHHHW consisting of five amino acid residues; the TMD gene (encoding human Spike-TMD) was then inserted (FIGS. 1 a-b).

Thereafter, the expression plasmid was used in E.coli recombinant expression system to induce expression of fusion protein by 0.5mM isopropyl-. Beta. -D-thiogalactoside (IPTG) (FIG. 1 c). The TrpLE-pentapeptide fusion protein is expressed in inclusion bodies of escherichia coli, and the TrpLE-pentapeptide fusion protein is purified by metal ion chelate chromatography to obtain the protein with higher yield and purity (figure 1 d). The flow chart of plasmid construction, expression, denaturation and purification is shown in FIG. 3.

2. Cutting condition optimization

Although the inclusion bodies are expressed and denatured, the protein with higher yield and purity is obtained. However, the expressed protein is still the structure of the fusion protein. How to efficiently remove TrpLE-pentapeptides is further contemplated.

The inventor has carried out optimization consideration of a plurality of experimental conditions, including a reaction system of a cleavage reaction, a cleavage temperature, a pH value, a time, selection of an organic phase or an aqueous phase or a buffer solution, and the like. Through extensive research analysis and experiments, it was determined that denaturants, detergents, cleavage solution pH, cleavage temperature and cleavage time should be significant for the cleavage reaction, which was optimized with great importance to the inventors.

The inventor surprisingly found that, in the reaction system of the cleavage reaction, the addition of a certain amount of guanidine hydrochloride is a key factor for realizing effective cleavage, if guanidine hydrochloride is not added, the fusion protein cannot be dissolved for cleavage, the cleavage efficiency of TrpLE is extremely low, and effective separation cannot be realized; if too much is added, the effect is not ideal.

The use of detergents is also necessary, and the inventors have found that cleavage efficiency is extremely low without the use of detergents, while some detergents may further reduce cleavage efficiency, and by comparison DPC is found to be relatively desirable.

Representative experimental results for different denaturant conditions (FIG. 2 a), different detergent conditions (FIG. 2 b), different cleavage solution pH (FIG. 2 c), cleavage temperature (FIG. 2 d) and cleavage time (FIG. 2 e) are presented in FIG. 2.

Considering the merits and merits of each cutting condition, the inventors finally determined the composition of the cut solution and the conditions as follows: 2M Guanidine hydrochloride (Guanidine-HCl), 25mM Tris, 150mM sodium chloride (NaCl), 100mM acetoxime, 1% Dodecyl Phosphatidylcholine (DPC), 2mM nickel chloride (NiCl) ₂ ) At a concentration, the pH of the cleavage solution was 8, the cleavage temperature was 37℃and the cleavage time was 24 hours.

Example 2 verification of the product after cleavage

The surface Spike protein Spike of SARS-CoV-2 (SARS 2) virus is a typical single transmembrane protein transmembrane domain, having an extracellular region, a transmembrane domain and an intracellular region. The transmembrane domain contains two methionine residues, which are subjected to artificial mutation in the original CNBr cleavage method. The present invention was expressed, cleaved and purified in the purification method described above by constructing the transmembrane segment of the Spike protein (Spike-TMD, 1194-1234) in the TrpLE-pentapeptide expression vector (fig. 4 a).

Molecular weight identification by mass spectrometry confirmed Ni ²⁺ Catalytic pentapeptide sequence cleavage occurs intermediate glycine and serine residues in the GSHHW sequence, with no side reactions occurring.

The purified protein was also identified to be of higher purity by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), as shown in FIG. 5a.

The results of size exclusion chromatography (size exclusion chromatography, SEC, also known as molecular sieves) also found that the corresponding 280nm absorbance peak of the protein exhibited sharp and symmetrical features, demonstrating that the state of the protein was uniform and stable, as shown in fig. 5a.

The sample is then used for ¹ H- ¹⁵ N TROSY (transverse relaxation-optimized spectroscopy) nuclear magnetic resonance detection as shown in FIG. 5b. TROSY results show that the chemical displacement signals of the samples are uniform, and the sample quality is good.

Example 3 preparation of T cell surface receptor beta chain (TCRbeta)

T cell surface receptor beta chains (TCRbeta) are also a typical single transmembrane protein transmembrane domain. The present invention was carried out by constructing TCR.beta. -TMD (131-178) in TrpLE-pentapeptide fusion expression vector (FIG. 4 b), and performing expression, cleavage and purification in the same purification manner as in example 1 above.

Further, the molecular sieve was combined to replace the micelle environment in which the transmembrane helix was located, and a sample of tcrp-TMD in DHPC solution was obtained. Mass spectrum identification and SDS-PAGE identification of the sample show that the molecular weight of the sample is correct and the purity is high (figure 6 a).

The sample was mixed with DMPC (final concentration DHPC: dmpc=0.3, lipid molecule to protein concentration ratio 10:1) and repeatedly snap frozen with liquid nitrogen, instant at 37 ℃ for a lot of timeAfter this time, a homogeneous bicell (bilayer membrane-like system) sample was obtained. The sample is used for nuclear magnetic resonance ¹ H- ¹⁵ N TROSY experiments. The TROSY results show that the chemical shift signal of the sample is uniform, and the sample quality is good, as shown in FIG. 6b.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims. All documents referred to in this application are incorporated by reference herein as if each was individually incorporated by reference.

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Claims (13)

1. A method for preparing a transmembrane domain of a single transmembrane protein, the method comprising:

(1) Creating an expression construct that expresses a fusion protein comprising the following elements in operative linkage: a transmembrane domain that facilitates expression of the tag-GSHHW pentapeptide-single transmembrane protein;

(2) Introducing the expression construct of (1) into a prokaryotic host cell for expression to obtain an expression and purification product;

(3) Cutting the transmembrane domain of the expression promoting label-GSHHW pentapeptide-single transmembrane protein, and separating to obtain the transmembrane domain of the single transmembrane protein; wherein the system for cutting contains guanidine hydrochloride, tris, sodium chloride, acetone oxime, dodecyl phosphatidylcholine and nickel chloride.

2. The method of claim 1, wherein the pro-expression tag is a tag that facilitates expression of the transmembrane domain of a single transmembrane protein in a prokaryotic host cell; preferably, the expression-promoting tag is TrpLE.

3. The method of claim 1, wherein the prokaryotic host cell is an e.

4. A method according to claim 3, wherein in step (2) the protein is expressed to form inclusion bodies followed by denaturation.

5. The method of claim 1, wherein in step (3), the system for cutting comprises:

guanidine hydrochloride: 2+ -1M; tris: 25.+ -.10 mM;

sodium chloride: 150.+ -.50 mM; acetone oxime: 100.+ -.30 mM;

dodecyl phosphatidylcholine: 1+/-0.5%; nickel chloride: 2.+ -. 1mM.

6. The method of claim 1, wherein in step (3), the cutting is performed by:

at a temperature of 37.+ -. 5 ℃, preferably 37.+ -. 3 ℃, more preferably 37.+ -. 2 ℃, more preferably 37.+ -. 1 ℃; or (b)

At a pH of 7.5 to 9.2, preferably 7.6 to 9.0, more preferably 7.7 to 8.5, more preferably 7.8 to 8.3, more preferably 7.9 to 8.2.

7. The method of claim 1, wherein the transmembrane domain of the single transmembrane protein comprises: transmembrane segment of Spike protein, transmembrane domain of T cell surface receptor β.

8. A recombinant expression vector comprising an expression construct comprising, operably linked: the expression promoting tag coding gene-GSHHW pentapeptide coding gene-single transmembrane protein transmembrane domain coding gene; preferably, the expression-promoting tag is TrpLE.

9. A prokaryotic host cell comprising an expression construct comprising the following elements operably linked: the expression promoting tag coding gene-GSHHW pentapeptide coding gene-single transmembrane protein transmembrane domain coding gene; preferably, the prokaryotic host cell is an E.coli cell.

10. Use of the recombinant expression vector of claim 8 or the prokaryotic host cell of claim 9 for recombinant expression of the transmembrane domain of a single transmembrane protein.

11. A kit for recombinant expression of a transmembrane domain of a single transmembrane protein, comprising:

the recombinant expression vector of claim 8;

a prokaryotic host cell; and

the cutting system comprises guanidine hydrochloride, tris, sodium chloride, acetone oxime, dodecyl phosphatidylcholine and nickel chloride.

12. A kit for recombinant expression of a transmembrane domain of a single transmembrane protein, comprising:

the prokaryotic host cell of claim 9; and

the cutting system comprises guanidine hydrochloride, tris, sodium chloride, acetone oxime, dodecyl phosphatidylcholine and nickel chloride.

13. The kit of claim 12, further comprising: IPTG, inclusion body denaturation liquid, triton X-100, nacl, ni column and packing thereof, imidazole, elution buffer, culture medium.

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