Fusion protein has two different meanings. One is obtained by DNA recombination technologygeneThe other is to mediate twoCell plasma membraneA group of fused proteins, as inSendaiVirusesLipid bilayerTwo kinds contained in lateral lobuleglycoproteinOne is to mediate the fusion between the viral envelope and the plasma membrane of the host cell.Another glycoprotein isHemagglutininCeramidase。 Two different proteins can be linked into one macromolecule by chemical methods or gene fusion.
In gene manipulation, for some small molecular numberspolypeptideGenes are often linked to a gene (such as lac) by fusion, and an enzyme (such asthrombin）To increase the stability of the product after expression in vivo, some deliberately make two molecules tandem fusion to improve the efficacy, such as IL-3 and GM-CSF.There are alsosignal peptidegeneformfusion geneTo secrete the expression product out of the membrane or cell.
Fusion protein technology is a purposeful method of gene fusion and protein expression to obtain a large number of standard fusion proteins. Using fusion protein technology, new target proteins with multiple functions can be constructed and expressed.
Prokaryotic expressionThe system is characterized by short duration and low cost, and is the main tool in scientific research.Its disadvantage is that the expression of eukaryotic protein has not been precisely modified;A large amount of protein is often precipitated into insoluble inclusion polymer, which requires complex denaturation andRenaturationProcess;It is difficult to secrete a large amount of protein.Eukaryotic expression system is characterized by proteinPost translation processingThere are many opportunities to be transformed into human source;Eukaryotic cellIt is easy to be transfected and has genetic stability and repeatability;The product can be secreted, and the purification is simple and the cost is low.
Structural design
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The basic method of constructing fusion protein is to modularize the natural or artificially coded polypeptide sequence with specific function, and synthesize it using the DNA sequence template encoded by gene, and then convert the first proteinTermination codonDelete and then connect the second protein gene with the termination codon to realize the co expression of the two genes.By controlling the exact position and density of each functional peptide module in the overall protein material, people can change the composition of fusion protein according to actual needs.
The design of fusion protein can be roughly divided into repeated structure based, growth factor based andCell adhesion moleculeCategory 3.The most typical type 1 fusion protein is the fusion protein from elastin like polymers (ELPs) and silk like polymers (SLPs).ELPs are extracellular matrix proteins composed of several repeated amino acid sequences, which are composed of two short peptide segments arranged alternately, mainly including VPGZG, VPGVG, APGVGV, VPGFGCGAG and VPGG.The most famous is VPGZG fusion protein (Z can be replaced by any amino acid except proline).The repeated VPGVG sequence can make the fusion protein have temperature sensitive characteristics, and the combination of temperature sensitive ELP and chemical, enzymatic, physical and other crosslinked hydrogels can generate a variety of applications.ELPs are mainly composed of glycine, alanine and serine, which are easy to form a reverse parallel β - folded lamellar structure, have multiple side chain chemical modification sites, good thermal stability and mechanical properties, and are commonly used in biomedicineSurgical suture , artificial skin and cartilage repair materials.For such fusion proteins, their sensitivity to temperature, pH and ionic strength can be precisely controlled by adjusting their amino acid sequence and molecular chain length, so as to achieve reversible dissolution transformation.[1]
Preparation method
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Most of the fusion proteins based on repetitive structure are short peptides, which do not have complex spatial structure. Therefore, only a simple peptide synthesis process can be used to obtain the target protein.By singleAmino acid synthesisPolypeptides are mainly realized by dehydration between two amino acids to form peptide bonds, which mainly includes the following basic steps: first, amino or carboxyl groups of amino acids with zwitterionic structure are protected accordingly, and then carboxyl groups are activated toActive intermediateAfter the coupling process is completed, the protective groups of amino acids on the peptide chain shall be selectively removed or completely removed in corresponding ways.At present, the commonly used peptide synthesis methods are liquid phase synthesis and solid phase synthesis.The liquid phase synthesis method is to prepare the required amino acid into a solution, protect the amino group of one amino acid, the carboxyl group of the other amino acids and the side chain group that does not participate in the reaction with chemical group protection, activate only the carboxyl group of the amino acid that participates in the reaction, and separate and remove the raw materials and activators that do not participate in the reaction after the completion of the coupling reaction to obtain the purified polypeptide product.Although the operation of this method is tedious, it has the advantage of high purity of the target product.In the solid phase synthesis method, the carboxyl group of the first amino acid at the C-end of the polypeptide sequence is fixed on the insoluble resin through esterification, and then the amino acid is used as the amino component to remove its amino protection group and couple with the excess activated carboxyl group to form peptide bonds. Repeated deprotection, condensation, and washing processes continue to synthesize to obtain the required peptide chain.Try again after synthesisTrifluoroacetic acidCut the product from the resin, remove all protective groups at the same time, purify and recover to obtain the required polypeptide.
In contrast, the synthesis process of the other two fusion proteins with certain spatial structure and complex sequence is much more complex.Accurate construction of the required DNA template is the basis for ensuring the diversity and stability of fusion proteins.After obtaining the target DNA sequence, connect it into a specific vector to obtain sufficient fusion protein particles.Due to the degeneracy of genetic genes, the same fusion protein can correspond to multiple DNA sequences, but the efficiency of sequence synthesis protein is related to the host species selected during amplification（Escherichia coliYeast and eukaryotic cells).For example, if the "AAG and AGA" fragments appear in the plasmids that need to use the Escherichia coli expression system, the protein yield will be greatly affected.Codon bias, tRNA availability, mRNA stability and mRNA structure all play an important role in gene expression. In addition, when selecting DNA fragments, we should try to avoid the existence of highly repetitive parts in the recombinant gene vector to avoid mismatch and affect mRNA transcription.
The specific steps are:
1. ProceedTarget geneCloning of: according to gene sequenceprinciple of complementarity, appropriately designedprimerUsing cDNA as template, different target DNA fragments were amplified by PCR.
2. Recombination in the carrier: byrestriction endonuclease The two DNA fragments were digested and recovered, and thenLigaseTwo will have the same endRestriction siteAnd cloned into high expressionPlasmid vectorMedium, buildRecombinant plasmid。
3. Will reorganizeexpression vector transfectionhost cellSelection markers were used for screening and sequencing.
4. Induced expression of fusion gene andExpression proteinPurification of.[1]
Key points of operation
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In the construction of fusion protein, a key problem is theConnector sequence(Linker), namely connecting peptide.Its length is very important for protein folding and stability.If the connector sequence is too short, it may affect the folding of the high-level structure of the two proteins, thus interfering with each other;If the splice sequence is too long, immunogenicity is involved, because the splice sequence itself is a new antigen.
Generally speaking, the linker with 3-5 amino acids can meet the requirements for correct folding of most fusion proteins.Some people tried to add a long peptide chain with hydrophobicity and certain extensibility between the fusion proteins, such as (Gly4Ser1), in order to separate the two, so as to alleviate mutual interference, and achieved satisfactory results.However, specific analysis is required when each protein is involved.When weConstruction of fusion proteinSeveral fusion modes should be selected to optimize the ideal connection mode.In addition, a large number of studies have shown that the flexibility and hydrophobicity of the linker peptide do not disturb the function of the proteinDomainIt is very important.
Unfortunately, there is no reliable selection criteria for the design of linker peptide sequences.At present, the design and selection of most linker peptide sequences still rely mainly on intuition.Although it has made great progress to rely on the primary structure of protein to predict its secondary structure, our understanding of the relationship between sequence and structure is still limited.
Clinical application
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1. DNA vaccine
At present,vaccineIt has gone through three generations: the first generation vaccine uses attenuated or killed pathogens to activate the bodyimmune system；The second generation vaccine is a component vaccine developed by biotechnology and recombinant DNA technology, which is injected into the body to induce immune response;The third generation vaccine is direct injectionGene recombinationTo activateHuman immune systemDNA vaccine.
Compared with traditional vaccines, DNA vaccines have obvious advantages, such as easy production, strong stability, low cost, etc., and can induce both humoral and cellular immune responses.
At present, the multivalent recombinant antibody fusion protein CYF196 (not yet listed in China) successfully produced by genetic engineering is a DNA vaccine, which can effectively prevent and treatlower respiratory tract infectionAnd asthma.Because CYF196 is the main receptor of the virus -Intercellular adhesion moleculeIt has high affinity and strong defense function against rhinovirus infection.
2. Bifunctional enzyme
Previous studies have found thatGene fusionAmong the large enzyme molecules constructed, if the entire coding sequence of each enzyme molecule used to form the fusion protein is retained in the new enzyme molecule, the fusion protein generally retains the respective enzyme activity of the enzyme molecule formed.
It is also found that in these newly constructed fusion proteins, the correct folding of the protein and the active site of each enzyme are not affected. Compared with a single enzyme, the specific activity of the enzyme of the fusion protein is 50% - 100%. For two or more enzymes that catalyze continuous reaction, the fusion protein formed by gene fusion can produce“Proximity effect” （proximity effect）。
The current research found that,β - galactosidase-Under certain conditions, the galactose dehydrogenase fusion proteincoupling reactionThe rate of NADH production is the same as that of the two enzymesreaction rate More than twice of.At the same time, the transition time is nearly four times shorter.
3. Targeted drug
Targeted drugs generally consist of two parts: one is drugs;The other part is the ligand that can specifically bind to the lesion.The two parts are fused together by fusion protein technology to form a uniqueconformationAnd functional proteins.
Gene fusion technology was first used in bacteria (mainlyEscherichia coli）For foreign genes (especially eukaryotic genes), the fusion expression method has many advantages. First, because foreign genes are generally connected to the C-terminal coding sequence of fusion proteins, there is no need to design another SD sequence,The existence of N-terminal fusion genes makes the expression of foreign genes relatively easy.Second, the expression products of foreign genes are oftenhost cellWhen the foreign gene is expressed in the form of fusion protein with a part of the coding sequence of the host's own protein, such as β - galactosidase, the degradation of the product by the host cell is often reduced.[2]
immunoglobulinFusion protein refers togeneLevel willTarget geneIt is linked to some Ig fragment genes andProkaryotic cellThe above two parts are expressed inDomainOfRecombinant protein。According to the connection between the target protein and different segments of Ig, it can be divided into two categories: one is Fab (Fv) fusion protein;The other is Fc fusion protein.
parathyroid hormoneParathyroid Hormone (PTH) is a single chain polypeptide hormone synthesized and secreted by the main cells of the parathyroid gland. Mature PTH contains 84 amino acid residues with a molecular weight of about 9500. It is one of the most important hormones regulating calcium and phosphorus metabolism and bone turnover in the human body.It mainly acts on the target cell membraneAdenylate cyclaseSystem, increase cAMP andPyrophosphate(PPi).
Cytokine recombinant fusion protein is a kind of fusion protein that uses genetic engineering methods to connect gene sequences encoding cytokines and other protein molecules with specific functions and express corresponding protein fusion products.Its structural feature is to fuse the functional active domain of cytokines with the active domain of other molecules, and each component can play a synergistic role, so that the biological activity of the fusion protein is greatly enhanced compared with each monomer.[3]
