35results about How to "Improve secretion efficiency" patented technology

The present invention provides novel purified and isolated nucleic acid sequences encoding procoagulant-active FVIII proteins. The nucleic acid sequences of the present invention encode amino acid sequences corresponding to known human FVIII sequences, wherein residue Phe309 is mutated. The nucleic acid sequences of the present invention also encode amino acid sequences corresponding to known human FVIII sequences, wherein the APC cleavage sites, Arg336 and Ile562, are mutated. The nucleic acid sequences of the present invention further encode amino acid sequences corresponding to known human FVIII sequences, wherein the B-domain is deleted, the von Willebrand factor binding site is deleted, a thrombin cleavage site is mutated, an amino acid sequence spacer is inserted between the A2- and A3-domains. Methods of producing the FVIII proteins of the invention, nucleotide sequences encoding such proteins, pharmaceutical compositions containing the nucleotide sequences or proteins, as well as methods of treating patients suffering from hemophilia, are also provided.

This invention relates to an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen protein or peptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked. More particularly, it relates to the method of eliciting an immune response directed against an antigen in a mammal comprising the steps of introducing the expression vector into a cell, expressing the vector to produce an antigen under conditions wherein the antigen is secreted from the cell, endocytosing the secreted antigen into the cell, processing the antigen, and presenting fragments to a receptor to elicit a T-cell response. In addition, this invention relates to a vaccine and a method of use. The invention also relates to the method of identifying MHC-II restricted epitopes.

This invention relates to an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen protein or peptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked. More particularly, it relates to the method of eliciting an immune response directed against an antigen in a mammal comprising the steps of introducing the expression vector into a cell, expressing the vector to produce an antigen under conditions wherein the antigen is secreted from the cell, endocytosing the secreted antigen into the cell, processing the antigen, and presenting fragments to a receptor to elicit a T-cell response. In addition, this invention relates to a vaccine and a method of use. The invention also relates to the method of identifying MHC-II restricted epitopes.

The invention discloses recombinant pichia pastoris for heterogenous efficient expression of lipase and application of the recombinant pichia pastoris. The recombinant pichia pastoris is obtained by converting plasmid HAC1-pPIC3.5K of an overexpression HAC1 gene in recombinant pichia pastoris X-33 which contains a pro-rml gene of a copy number 4 and is capable of expressing Pro-RML. When the strain is subjected to 96 hours of flask shaking fermentation, the extracellular enzyme activity is up to 1078U / mL at most, and the enzyme activity secretion efficiency is up to 47U / OD600. 120 hours of fermentation is needed when pichia pastoris which is disclosed by patent CN103361327A and has a rhizomucor mieheilipase copy number of 2 meets the highest extracellular enzyme activity and enzyme secretion efficiency, the extracellular enzyme activity is 1038U / mL at most, and the enzyme activity secretion efficiency is only 25U / OD600. By adopting the recombinant pichia pastoris, expression of rhizomucor mieheilipase is effectively promoted, on the premise that the highest extracellular enzyme activity is not influenced, the secretion efficiency of the rhizomucor mieheilipase is increased by 1.9 times, and the fermentation time is shortened by 24 hours.

The invention belongs to the field of protein engineering and genetic engineering, and relates to a small peptide strengthening mold sequence of a strong secretory signal peptide and an application thereof. The small peptide strengthening mold sequence comprises an amino acid sequence shown in the general formula: M(AlphaXBetaYGamma / AlphaYBetaXGamma)n, wherein the X represents an acidic amino acid; the Y represents a basic amino acid; the Alpha represents 0 to 2 neutral amino acids; the Beta represents 0 to 2 neutral amino acids; the Gamma represents 1 to 10 neutral amino acids; and the n represents 1 to 3. According to the application of the small peptide strengthening mold sequence, the small peptide strengthening mold sequence is use for constructing a carrier for strengthening the secretion capability of a common signal peptide, thereby being a method for improving the common signal peptide for the secretory expression of a foreign protein.

The present invention provides novel purified and isolated nucleic acid sequences encoding procoagulant-active FVIII proteins. The nucleic acid sequences of the present invention encode amino acid sequences corresponding to known human FVIII sequences, wherein residue Phe309 is mutated. The nucleic acid sequences of the present invention also encode amino acid sequences corresponding to known human FVIII sequences, wherein the APC cleavage sites, Arg336 and Ile562, are mutated. The nucleic acid sequences of the present invention further encode amino acid sequences corresponding to known human FVIII sequences, wherein the B-domain is deleted, the von Willebrand factor binding site is deleted, a thrombin cleavage site is mutated, an amino acid sequence spacer is inserted between the A2- and A3-domains. Methods of producing the FVIII proteins of the invention, nucleotide sequences encoding such proteins, pharmaceutical compositions containing the nucleotide sequences or proteins, as well as methods of treating patients suffering from hemophilia, are also provided.

The invention discloses a heat-resistant neutral pullulanase mutant and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, amino acids at the 73th site, the 135th / 314th site, the 135th / 314th / 126th site and the 135th / 314th / 126th / 72nd site of pullulanase are respectively mutated, so that four pullulanase mutants with improved thermal stability and unchanged or improved catalytic efficiency are obtained. At the temperature of 70 DEG C, the half-life periods of the four mutants are respectively 2.6, 1.9, 3.3 and 3.1 times that of a wild type; and the Tm values are respectively increased by 7.0 DEG C, 6.5 DEG C, 6.7 DEG C and 6.9 DEG C. The extracellular enzyme activity of pullulanase reaches 9.7 U / mL by fusing OptPelB at the N terminal of the high-temperature-resistant neutral pullulanase mutant, and is 4.9 times that of a wild type. The heat-resistant neutral pullulanase mutant capable of secreting and expressing has the application potential of high-value utilization of starch raw materials.

The invention discloses a method for increasing the secretion efficiency of bile salt hydrolase, belonging to the technical field of genetic engineering. In the invention, by adopting the recombinant DNA technology, the bile salt hydrolase gene bsh from lactobacillus plantarum is fused with signal peptide alpha-MF and connected with a carrier pPIC9K and transformed into pichia pastoris GS115 to obtain bile salt hydrolase producing recombinant pichia pastoris. By exogenously adding castanospermine and Fe<2+>, the secretion efficiency of pichia pastoris bile salt hydrolase is increased, the activity of the fermentation 3d bile salt hydrolase reaches 14.96U / mL which is 1.74 times higher than that without exogenous substance.

Provided is a method for improving secretion efficiency of a recombinant foreign protein in a yeast expression system. The method includes transforming a yeast host with a recombinant foreign gene construct containing a galactose-inducible promoter, a secretion signal sequence and a gene encoding the foreign protein to construct a transformed yeast strain; and culturing the transformed yeast strain under the condition that the activity of the galactose-inducible promoter is controlled. Improved secretion efficiency of the foreign protein can be achieved by decreasing overexpression-induced insoluble precipitation of the recombinant foreign protein suffered by a conventional galactose-inducible promoter-based yeast expression system, via appropriate control of a level of galactose functioning as an inducer of the galactose-inducible promoter in cells. Due to improved secretion efficiency of the recombinant foreign protein, present invention makes a contribution to improvement in productivity of recombinant foreign proteins in the yeast expression system and reduction in production costs.

A method of identifying nucleotide sequences coding for signal peptides in lactic acid bacteria, using a DNA molecule comprising a transposon including a promoterless reporter gene from which DNA molecule a region between the LR and the reporter gene is deleted and the DNA molecule comprises a DNA sequence coding for a secretion reporter molecule. By deleting the region between the LR and the reporter gene, stop codons in-frame with the secretion reporter molecule is removed which upon transposition permits translational fusions from upstream the LR.

A method for improving the secretion rate of exogenous protein expressed by silkworm cells, designing and synthesizing an RNAi interference fragment for interfering with the BiP gene in silkworm BmN cells, transfecting BmN cells for 24 hours and then continuing to transfect the expression vector carrying the foreign gene, after 72 hours That is, the secretion rate of the foreign protein can be significantly increased. This method is simple and easy to operate, and can effectively improve the secretion efficiency of exogenous proteins expressed in silkworm cells. It can provide a new idea to improve the expression of membrane proteins by using BiP etc. as molecular targets, and is conducive to in-depth understanding of baculovirus Expression system, and provide a reference for the mechanism research of signal peptide involved in protein secretion.

The invention provides a method for improving recombinant protein secretion efficiency, which is characterized in that a secretion signal sequence SS in the homeotic allotype domain of the mouse transcription factor Engrailed 2 is fused to the N-terminal of the recombinant protein; wherein the amino acid sequence of the secretion signal sequence SS is that: Ala Gln Glu Leu Ser Leu Asn Glu Ser Gln. The invention has the advantages that: the secretion efficiency of human catalase using the invention is improved to 2.3 times than the normal secretion efficiency guided by bovine Beta-casein signal peptide and provides an effective solution for improving recombinant protein secretion efficiency in industrial production.

The invention discloses a signal peptide capable of improving secretion efficiency. A nucleotide sequence is shown in 1) or 2): 1) the nucleotide sequence is shown as SEQ ID NO.1; 2) after transforming the SEQ ID NO.1 sequence, the nucleotide sequence still performs the function of signal peptide and the secretion efficiency of the nucleotide sequence does not have fundamental change. The invention provides a signal peptide capable of improving the secretion efficiency, the secretion capability of strains can be remarkably improved after the signal peptide is used, and the enzyme activity of asparaginase can be improved by 1.5 times. The enzyme production capability of the transformed strains is remarkably improved, and the transformed strains are more suitable for industrial application, the production cost can be lowered, and the production efficiency can be improved.

The invention relates to a method for the production and non-covalent surface display of antibodies and derived fragments as well as molecule libraries based thereon on the surface of S. cerevisiae cells. The non-covalent manner of the surface display renders possible the selection of specific variants by means of high throughput screening and the subsequent switchable secretion of the selected binding molecule into the culture supernatant for biochemical characterization.

Provided is a method for improving secretion efficiency of a recombinant foreign protein in a yeast expression system. The method comprises transforming a yeast host with a recombinant foreign gene construct comprising a galactose-inducible promoter, a secretion signal sequence and a gene encoding the foreign protein to construct a transformed yeast strain; and culturing the transformed yeast strain under the condition that the activity of the galactose-inducible promoter is controlled. Improved secretion efficiency of the foreign protein can be achieved by decreasing over-expression-induced insoluble precipitation of the recombinant foreign protein suffered by a conventional galactose-inducible promoter-based yeast expression system, via appropriate control of a level of galactose functioning as an inducer of the galactose-inducible promoter in cells. Due to improved secretion efficiency of the recombinant foreign protein, present invention makes a contribution to improvement in productivity of recombinant foreign proteins in the yeast expression system and reduction in production costs.

The invention discloses a signal peptide for optimizing high-level secretory expression of keratinase Ker and application of the signal peptide and belongs to the fields of genetic engineering and enzyme engineering. The signal peptide is characterized in that 46 secretory signal peptides from bacillus subtilis are preliminarily screened and 6 secretory signal peptides capable of obviously improving the secretion volume of the keratinase in the 46 secretory signal peptides are directionally transformed, so the signal peptide for optimizing the high-level secretory expression of keratinase is obtained. An N terminal of the signal peptide is provided with first nine amino acid residues of a signal peptide AbnA; an H terminal of the signal peptide is provided with a hydrophobic sequence of a signal peptide YfkN, and a C terminal of the signal peptide is provided with last four amino acid residues of a signal peptide PhoB. The signal peptide disclosed by the invention is fused at the N terminal of the keratinase Ker, so the secretion efficiency of the keratinase of recombinant bacillus subtilis is remarkably improved, and the enzyme activity of extracellular keratinase of the bacillus subtilis is improved by 3.39 times. The extracellular keratinase producing capability of a transformed strain is remarkably improved, the industrial application is better facilitated, the production cost can be reduced, and the production efficiency is improved.

The invention discloses a signal peptide for mediating protein secretion expression and belongs to the field of protein engineering and gene engineering. By modifying known signal peptides PelB and OmpA, the N terminal of the signal peptide contains the first three amino acid residues MKK of the signal peptide OmpA, the C terminal of the signal peptide contains the last three amino acid residues AQA of the signal peptide OmpA, and a middle hydrophobic structure is the hydrophobic sequence LLPTAAAGLLLLAAQP of the signal peptide PelB. After the signal peptide capable of effectively improving protein secretion efficiency is used, the secretion expression capability of a strain on target protein is obviously improved; the extracellular enzyme activity of phospholipase D of target protein can be improved by 2 times, the extracellular enzyme production capability of the modified strain is significantly improved, and industrialized application is better facilitated.

The invention belongs to the field of protein engineering and genetic engineering, and relates to a class of strongly secreted signal peptide enhancing small peptide modules and applications thereof. The strong secretory signal peptide enhancing small peptide motif of the present invention has an amino acid sequence of the following general formula: M(αXβYγ / αYβXγ)n, wherein X represents an acidic amino acid; Y represents a basic amino acid; α is 0-2 β represents 0-2 neutral amino acids; γ represents 1-10 neutral amino acids; n is 1-3. The application of the strong secretory signal peptide enhancing small peptide motif of the present invention is to use the strong secretory signal peptide enhancing small peptide motif of the present invention to construct a carrier that enhances the secretory ability of ordinary signal peptides, so as to improve It secretes a method of expressing a foreign protein.

The invention discloses recombinant pichia pastoris for heterologously and efficiently expressing lipase and application of the recombinant pichia pastoris. The recombinant pichia pastoris is obtained through converting plasmid SEC31-pPIC3.5K overexpressing a gene SEC31 in recombinant pichia pastoris X-33, which contains a 4-copy number pro-rml gene and can express Pro-RML. According to the recombinant pichia pastoris and the application thereof, the expression of the rhizomucor miehei lipase is effectively promoted, the efficiency of secretion of the lipase is increased, the enzyme activity reaches 996U / mL after 144 hours of shake-flask fermentation, and the strain ectoenzyme activity secretion efficiency reaches 38U / OD600 and is higher than that (26U / OD600) of two recombinant pichia pastoris strains of rhizomucor miehei lipase copy numbers concerned in the patent CN103361327A.