200results about "Fusions for enhanced expression stability/folding" patented technology

The invention relates to the technical field of gene engineering, in particular to Escherichia coli for efficient soluble expression of recombinant human collagen III and construction and application thereof. The invention constructs Escherichia coli for efficient soluble expression of recombinant human III type collagen, and the Escherichia coli can co-express the recombinant human III type collagen and molecular chaperone which are regulated and expressed by signal peptide. Experiments prove that the engineering bacterium provided by the invention has the capability of efficient soluble expression of the recombinant human III-type collagen, and the recombinant human III-type collagen provided by the invention has good stability and anti-inflammatory effect.

Provided are a recombinant human interleukin-11 mutant, and a conjugate of the mutant conjugated to a chemical molecule and a use thereof. The recombinant human interleukin-11 mutant achieves soluble expression in an Escherichia coli expression system by means of an SUMO sequence tandem fusion method. The conjugate is obtained by the site-specific conjugation of the mutant to the chemical molecule at a mutant amino acid residue in position W147 by means of a covalent bond. The chemical molecule is at least one of polyethylene glycol, an alkanoic acid aliphatic chain, and a hydrophilic high-molecular polymer containing an alkanoic acid aliphatic chain. The conjugate retains the binding capacity to human interleukin-11 receptor A while demonstrating significantly reduced binding capacity to human IL-6 signal transducer receptor. The conjugate shows the ability to block the signaling between human IL-11 and IL-11 RA and between human IL-11 and GP130 either in vivo or in vitro and can be used for the treatment of associated diseases with tissue fibrosis as a clinical manifestation caused by elevated primary or secondary IL-11.

The invention discloses a method for improving the solubility and thermal stability of sweet protein, and belongs to the technical field of biosynthesis, the method comprises the following steps: constructing a gene tandem recombinant plasmid containing a monellin x-3C-sfGFP-3C-monellin y expression cassette, x is greater than or equal to 1, y is greater than or equal to 1, 5 is greater than or equal to x + y is greater than or equal to 3, a 3C protease recognition sequence is also inserted between adjacent copies of monellin, and each monellin is connected with a purification tag; the gene tandem recombinant plasmid is transformed into escherichia coli and inducible expression is carried out, thalli are split and purified to obtain fusion protein, 3C protease is used for enzyme digestion, and the target protein is obtained after re-purification. The total protein yield and the solubility proportion are remarkably improved by adopting a series construction mode of plasmids, meanwhile, the stability of monellin is effectively improved, the purification step is simpler and more convenient, and the method is suitable for industrial production.

The invention discloses recombinant yarrowia lipolytica with high yield of 3-fucosyllactose as well as a construction method and application of the recombinant yarrowia lipolytica. According to the invention, a reinforced lactose transporter lac12 gene, a reinforced GDP-mannose-4, 6-dehydratase gmd gene, a reinforced GDP-L-fucose synthase gmer gene and a 3-fucosyltransferase SUMO-fut3Bc gene with a reinforced SUMO tag are subjected to integrated expression on a recombinant yarrowia lipolytica chromosome, and a 6-phosphogluconolactonase pgl gene is subjected to overexpression, so that a recombinant yarrowia lipolytica strain is obtained. The constructed recombinant yarrowia lipolytica can maintain the genetic stability of a target gene, the yield and production efficiency of 3-fucosyllactose are remarkably improved, and the requirements of industrial production can be met.

Provided are a GLP-1GIP analog, an FGF21 protein, a trifunctional protein, and use thereof. The trifunctional protein comprises the GLP-1 / GIP analog and the FGF21 protein. Further provided is use of the GLP-1 / GIP analog, the FGF21 protein, and the trifunctional protein in the preparation of a drug for preventing and / or treating diseases related to GLP-1, GIP, and / or FGF21. The provided GLP-1 / GIP analog, FGF21 protein, and trifunctional protein can synergistically regulate blood glucose and lipid levels in vivo, meet multiple needs in patients with type 2 diabetes, including lowering blood glucose, alleviating hepatic steatosis, promoting weight loss, ameliorating circulating lipid metabolism disorder, etc., and have better biological safety and tolerability and longer in vivo half-lives.

Provided are a method for constructing a soluble expression plasmid mutant library, and the use thereof. The method for constructing the soluble expression plasmid mutant library comprises: introducing mutations into a ribosome binding site on a soluble expression plasmid, a sequence composition and length of a region between the ribosome binding site and an initiation codon of a fusion protein to be expressed, and a translation initiation region, so as to obtain a mutant library. By means of the method, plasmid mutants with increased expression levels are obtained, thereby increasing the expression level of a target fusion protein and the final yield of a target polypeptide. The present invention solves the problem of low soluble expression levels of certain polypeptides in Escherichia coli.

The present invention relates to research tools for structural biology, particularly for facilitating the determination of three-dimensional structures of biological macromolecules. More specifically, the present invention helps to improve the overall feasibility of structure determination, resulting in higher resolution and better quality of three-dimensional structures of proteins through complex formation with novel fusion polypeptides.

The present disclosure relates generally to recombinant polynucleotides comprising novel pro-region DNA sequences. Certain aspects of the disclosure relate to recombinant Gram-positive bacterial strains comprising one or more introduced polynucleotides comprising novel pro-region DNA sequences operably linked to a DNA sequence encoding a protein of interest.

This invention discloses a functional peptide for increasing recombinant protein yield, the sequence of which is shown in any of SEQ ID No. 1-15. The encoding gene and its applications are also disclosed, along with corresponding methods for increasing protein yield. The functional peptide of this invention can significantly increase the yield of recombinant proteins, significantly reduce the loss rate of recombinant proteins during expression and purification in the MtuΔI-CM protein expression system, reduce the production cost of recombinant proteins, and improve the stability of the target protein. It is suitable for the production of various recombinant human proteins, laying the foundation for large-scale, low-cost mass production of various recombinant proteins and possessing good commercial application prospects.

The present invention provides a method of modulating a molecular mass of a fusion polypeptide, wherein the fusion polypeptide comprises a target polypeptide fused to a tag suitable for affinity purification or detection of the target polypeptide, wherein the tag comprises at least one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof and wherein the molecular mass and / or an isoelectric point of the fusion polypeptide is modulated by increasing the molecular mass of the tag via combining the one constant domain of an immunoglobulin light chain or orthologous polypeptide or fragment thereof with one or more further constant domain(s) of an immunoglobulin light chain or orthologous polypeptide(s) or fragment(s) thereof; wherein the constant domain of the immunoglobulin light chain or orthologous polypeptide or fragment thereof of the tag is monomeric; and wherein optionally the target polypeptide is fused to the tag via a first linker. The invention further provides a fusion polypeptide obtained or obtainable by this method. The invention also provides a tag and a fusion polypeptide comprising the tag.

Provided herein are compositions and methods for adoptive cell therapy comprising engineered immune cells that express a tumor antigen-targeted chimeric antigen receptor and a prodrug converting enzyme.

Disclosed are compositions and methods for treating a disease or disorder such as cancer in a subject in need thereof. In some embodiments, the methods include administering to the subject a vector that has a first nucleic acid sequence encoding a promoter operably linked to each of a second nucleic acid sequence encoding a therapeutic polypeptide, and a third nucleic acid sequence encoding a peptide domain that is stabilized when phosphorylated by kinase activity in a target cell and / or tissue. In some embodiments, the target cell and / or tissue can be a cell and / or tissue undergoing a stress response. In some embodiments, the target cell and / or tissue can be a cell and / or tissue in which a CK2 kinase is active. The kinase activity can be elevated extracellular regulated kinase (ERK) activity, p38 MAP kinase activity, and / or CK2 activity.

The application discloses a method for conformational signal peptide-mediated secretion and expression of heterologous proteins in Escherichia coli, and specifically comprises the following steps: simultaneously fusing at least two heterologous proteins to a conformational signal peptide (such as sfGFP) and constructing a heterologous recombinant protein expression vector, wherein the conformational signal peptide has a beta-barrel structure, and the fusion sites are at least two of the amino terminal end, the carboxyl terminal end and a plurality of loop rings of the conformational signal peptide; and then transforming the heterologous recombinant protein expression vector into Escherichia coli and culturing the obtained genetic engineering strain for expression. According to the method, the extracellular secretion and expression of the heterologous proteins in the Escherichia coli can be realized without the mediation of a classical signal peptide, the sfGFP as a fusion tag has no influence on the function of the target protein, and the corresponding target protein can be quickly expressed and purified; and the method expands the secretion and expression path of the protein, can realize the simultaneous expression of a plurality of heterologous proteins, and can effectively improve the expression amount of the target protein.

The present invention provides a recombinant engineered bacterium for producing recombinant canine albumin and its application, relating to the field of biochemistry, specifically, genetic engineering technology. By integrating overexpression of one or more of the protein-folding-related genes PBN1, KAR2, and ERV2 into the strain while simultaneously expressing recombinant canine albumin, the present invention significantly improves canine albumin expression, with relative protein expression reaching up to 491% and specific yields reaching up to 32.83 g / L, demonstrating broad application prospects.

Provided is a polypeptide combination, which comprises a Rubisco polypeptide from a species of the family Chlamydomonadaceae, and a fusion polypeptide comprising an EPYC1 polypeptide of a species of the family Chlamydomonadaceae and a mineralizing peptide, wherein the mineralizing peptide can convert a metal-containing compound into a compound capable of converting light energy into chemical energy, such as a metal oxide. Further provided are a polynucleotide or a vector encoding the polypeptide combination, a microorganism containing the polypeptide combination, the polynucleotide or the vector, and a method of using the microorganism.

The invention relates to the technical field of biology, in particular to a recombinant pichia pastoris engineering bacterium, a construction method of the recombinant pichia pastoris engineering bacterium and application of the recombinant pichia pastoris engineering bacterium in secretory expression of ovotransferrin. The heterologous expression of ovotransferrin is effectively realized by optimizing ovotransferrin genes and utilizing molecular chaperone genes to strengthen the regulation and control capability of hosts, and meanwhile, by optimizing signal peptides and constructing high-copy recombinant pichia pastoris expression strains, the low-copy expression condition is verified and compared by utilizing fermentation expression, so that the heterologous expression of ovotransferrin is realized. According to the high-copy recombinant pichia pastoris engineering bacteria constructed by the invention, the ovotransferrin expression quantity is obviously improved and reaches 140 mg / L, the secretory expression efficiency of the ovotransferrin is effectively improved, and theoretical guidance is provided for heterologous expression and industrial production of the ovotransferrin.

The present disclosure provides a peptide tag, and a nucleic acid encoding the peptide tag. The peptide tag of the present disclosure can reduce an aggregation property of a protein in a cell. Specifically, the peptide tag of the present disclosure can be a peptide tag in which 5% or more and less than 45% of amino acids contained in an amino acid sequence thereof are acidic amino acids, and (b) 20% or more of the amino acids contained in the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A.

The present invention relates to polypeptides comprising a first amino acid sequence comprising one or more GG repeat sequences and a peptide or polypeptide of interest in form of a fusion protein that exhibits increased renaturation efficiency and optionally also improved expression. Also encompassed are nucleic acids encoding these polypeptides, host cells that comprise said nucleic acids, and methods for protein expression and renaturation using said nucleic acids, host cells and polypeptides.

Provided are a fusion protein of a lixisenatide precursor and the use thereof. The fusion protein comprises a lixisenatide precursor and a tag protein fused to the N-terminus of the lixisenatide precursor. The lixisenatide precursor has an amino acid sequence as set forth in SEQ ID NO: 1; and the tag protein is selected from any one of the following: a Fh8 tag protein, a Ffu209 tag protein, a CBM tag protein, a Sumo tag protein or a Trx tag protein. The lixisenatide precursor is fused with the tag protein, and the lixisenatide precursor with the tag protein has a relatively high expression level, and can be used to prepare a large number of high-purity lixisenatide precursors, thereby better conducting industrial production and application of lixisenatide.

The present invention relates to designed ankyrin repeat domains with binding specificity for serum albumin, in particular such designed ankyrin repeat domains with improved stability. The invention further relates to recombinant binding proteins comprising such designed ankyrin repeat domains, nucleic acids encoding such designed ankyrin repeat domains or proteins, pharmaceutical compositions comprising such proteins, and the use of such proteins or pharmaceutical compositions in the treatment of diseases.

The invention relates to construction of a proline hydroxylase alpha subunit mutant from a human placenta and an expression vector of the proline hydroxylase alpha subunit mutant, and a mutant protein which is efficiently expressed in Ecoli BL21 (DE3) and has greatly improved hydroxylation efficiency and a construction body of the mutant protein are obtained by analyzing and screening a natural hydroxylase subunit.

Disclosed herein are rare earth element (REE) sensors comprising an REE binding element and a fluorescent component, which can be expressed on a cell surface. Also disclosed are systems and methods of recovering rare earth elements from a sample, the method comprising exposing an aqueous solution comprising one or more rare earth elements. Also disclosed are methods of discovering new, useful REE binding elements.

The invention discloses a trimer motif and application of the trimer motif in expression of RSV (Respiratory Syndrome Virus) trimer protein, solves the problems of epitope shielding, low expression quantity and the like of a trimer protein vaccine, and realizes efficient and high-expression RSV trimer protein vaccine expression.

This invention belongs to the field of biomedicine and relates to the DHX8 fusion protein and its preparation method. The amino acid sequence of the DHX8 fusion protein is shown in SEQ ID NO:1. The method of this invention includes: 1) amplifying the nucleic acid sequence encoding the DHX8 fusion protein; 2) cloning the nucleic acid sequence into a prokaryotic expression vector; 3) transforming the prokaryotic expression vector into E. coli strain; 4) inducing the expression of the DHX8 fusion protein; and 5) purifying the DHX8 fusion protein. The DHX8 fusion protein of this invention has ATP hydrolase activity and helicase activity. The method of this invention is easy to mass-produce and can be used for high-throughput screening of DHX8 helicase inhibitors. Furthermore, its expression system is simple.

The present invention relates to coronavirus antigens comprising a coronavirus nucleocapsid specific amino acid sequence, compositions and kits comprising them and methods of production thereof. Further included are methods of using said coronavirus antigens for detecting anti-coronavirus antibodies in a sample, and methods of differentiating between immune responses in a patient caused by a natural coronavirus infection or by vaccination against coronavirus.

The invention provides compositions and methods for nucleic acid synthesis, including ordered and continuous complementary DNA (cDNA) synthesis across non-continuous templates using a modified eukaryotic non-long terminal repeat reverse transcriptase (non-LTR RT) protein.

The invention belongs to the technical field of globular protein synthesis, and particularly relates to a chaperonin-assisted recombinant human hFGF-2 protein preparation method and application thereof, the chaperonin-assisted recombinant human hFGF-2 protein preparation method comprises the following steps: S1, constructing a recombinant vector TrxA-6H-DDDDK-hFGF2 containing an hFGF-2 fusion protein coding sequence; s2, transforming the recombinant vector into an escherichia coli host cell for induced expression to obtain an expression thallus; s3, crushing the expression thalli to obtain a cell lysis supernatant; s4, taking the supernate, carrying out nickel column affinity chromatography, carrying out desalination treatment on the fusion protein by adopting gel chromatography, and collecting a fusion protein eluent; s5, performing enterokinase cutting treatment on the fusion protein to obtain an enzyme-digested product; s6, performing heparin affinity chromatography on the enzyme digestion product, and collecting hFGF-2 protein eluent; and S7, carrying out dialysis treatment on the protein eluent to obtain the recombinant hFGF-2 protein.

The invention belongs to the field of protein engineering. The invention provides high-stability non-hydroxylase modified recombinant humanized collagen as well as a preparation method and application thereof. The amino acid sequence of the high-stability non-hydroxylase modified recombinant humanized collagen comprises one of amino acid sequences shown as SEQ ID NO.1 or SEQ ID NO.2. The invention further provides a preparation method of the high-stability non-hydroxylase modified recombinant humanized collagen. Under the condition that the recombinant humanized collagen does not need to be subjected to hydroxylation modification, the triple helix stability is remarkably improved, a streptococcus Scl2 collagen-like protein triple helix stable structural domain is excavated through molecular dynamics simulation to be embedded with a natural collagen sequence, the natural sequence is reserved, the biological activity of the collagen is ensured, and the recombinant humanized collagen has the advantages of being simple in structure, convenient to use and the like. The dependence of a traditional recombinant expression technology on hydroxylation modification of proline is also avoided.

Disclosed are a thioredoxin mutant, preparation method thereof, and application thereof in production of recombinant fusion protein. The thioredoxin mutant is prepared by modifying at least one amino acid in the amino acid sequence of SEQ ID NO: 1.

The invention provides a VNP6-Sortase A7plus fusion protein as well as a preparation method and application thereof. The fusion protein comprises a VNP6 sequence and a Sortase A7plus sequence which are sequentially connected from an N end to a C end, the amino acid sequence obtained after the VNP6 sequence and the Sortase A7plus sequence are connected is as shown in SEQ ID NO: 5. According to the invention, a VNP6 tag is introduced to the N end of Sortase A7plus protein, and an optimized culture strategy is combined, and intracellular vesicles of escherichia coli are induced to be concentrated, so that the problem of molecular congestion caused by high expression load is effectively relieved, and the effect of increasing the expression quantity of Sortase A7plus is achieved.