767 results about "Pichia" patented technology

Methods are provided for the synthesis and secretion of recombinant proteins preferably large mammalian proteins or hetero-multimeric proteins at high levels and for prolonged time in polyploid, preferably diploid yeast. These methods use various mating competent yeast, including Pichia. In a preferred embodiment, a first expression vector is transformed into a first haploid cell; and a second expression vector is transformed into a second haploid cell. The transformed haploid cells, each individually synthesizing a non-identical polypeptide, are identified and then genetically crossed or fused. The resulting diploid strains are utilized to produce and secrete fully assembled and biologically functional hetero-multimeric protein.

The present invention intends to find out a gene participating in addition of mannose phosphate to a sugar chain of a glycoprotein originating in a yeast belonging to the genus Pichia and provide a means of controlling the same. The present invention also intends to provide a process for producing a protein with reduction of an acidic sugar chain by using the thus controlled yeast strain belonging to the genus Pichia. Namely, the present invention includes a protein participating in the addition of mannose phosphate to a sugar chain of a glycoprotein; a gene encoding this protein; a mutant of this gene; a vector carrying the mutant gene; a yeast strain belonging to the genus Pichia having been transformed by this vector; a process for producing a protein with reduction of an acidic sugar chain by using the transformed yeast strain; and a glycoprotein thus produced.

The invention discloses a method for synthesizing ethyl caproate under the catalysis of yeast display lipase. The method comprises two steps, namely the production of full cellular zymin and the synthesis of ethyl caproate. The method comprises the following: a step of cloning the lipase gene into the Pichia yeast surface display vector pKFS to construct the Pichia yeast surface display expression vector pKFS-lipase which is transformed into the host bacteria of Pichia yeast through linearization, a yeast genetic engineering bacteria capable of displaying active pheron on the surface of the Pichia yeast being obtained through screening, and the engineering bacteria being fermented in a rocking bottle to obtain a thallus which is used to make the full cellular zymin after 24 hours of vacuum freeze drying; and then a step of adopting caproic acid and alcohol as raw material which is esterified at the action of the biocatalyst of yeast full cellular zymin with the lipase displayed on the surface to obtain the ethyl caproate product. The method is capable of collecting thalli centrifugally for reuse after the reaction with short reaction time, high yield and good operational stability, thereby greatly reducing production costs.

The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris are also provided.

The invention discloses a preparation method of a recombinant porcine circovirus type 2 Cap antigen, which comprises the following steps: using a yeast expression system to amplify the PCV2-ORF2 gene; then, constructing an expression engineering bacterium: inserting an amplification sequence into a yeast expression vector to form a recombinant expression vector, linearizing, and transforming into a pichia pastoris host cell so as to finish the construction of the expression engineering bacterium; and finally, carrying out secretory expression on the recombinant microzyme to obtain the recombinant porcine circovirus type 2 Cap antigen protein. The yeast expression system can carry out processing, folding and posttranslational modification on the expressed protein, so that the expressed protein has biological activity. Compared with Escherichia coli, the yeast expression system has more advantages; and compared with a mammal cell expression system, the yeast expression system is more convenient to operate. Compared with Saccharomyces cerevisiae, the pichia pastoris can not be easily subjected to hyperglycosylation, thereby facilitating the separation and purification steps. By using the yeast expression system, the invention has the advantages of simple operation process, high expression quantity and low production cost, can implement large-scale production, and is beneficial to the popularization and application of the porcine circovirus subunit vaccine.

The invention discloses acid beta-mannase, genes, engineering bacteria and a structure thereof. A coding gene manAsp is from Aspergillus niger CBS 513.88, the coding gene has a nucleotide sequence expressed as SEQ ID NO.3 or 4, and the acid beta-mannase obtained by coding has an amino acid sequence expressed as SEQ ID NO.1 or 2. The structure of the engineering bacteria is obtained by guiding a pichia pastoris constitutive expression vector (pGAPZa-man) containing the coding gene sequence of the beta-mannase into pichia pastoris. The acid beta-mannase of the invention has the following properties: at the optimal pH of 5.0 and the optimal temperature of 40 DEG C, the beta-mannase has better stability when the pH is between 3.0 and 7.0, and the residual enzyme activity is 66.39 percent when the beta-mannase is treated for 1 hour at the temperature of 90 DEG C; the engineering bacteria achieve efficient expression of the acid beta-mannase; and the fermentation process is simple, has low extracting cost, is suitable for large-scale industrialized production, and has broad application prospect in the industries of feed, food, medicine, energy and the like.

The invention discloses a glucose oxidase mutant gene, expression and application thereof. In the invention, 272 basic groups are changed through codon optimization and GC content change and the content of GC is reduced to 48.44% from 55.54% so as to obtain the glucose oxidase mutant gene, wherein the basic group is represented by SEQ ID NO.2. The glucose oxidase mutant gene is transferred into pichia yeast to express; the experimental result shows that the secretory expression level of the glucose oxidase mutant gene in the pichia yeast is significantly improved by comparing with the same before mutation; compared with partial research at home and abroad, the final secretory expression of the glucose oxidase mutant gene achieves high expression level, thereby building the foundation for further expansion of industrial production. The determination of enzymatic properties of the glucose oxidase mutant gene shows that the recombinant glucose oxidase protein expressed by the glucose oxidase mutant gene has good thermal stability and high enzyme activity.

The present invention relates to genes of the Pichia pastoris expression codon-optimized main capsid protein L1 of human papilloma virus types 52,31 and 45, a vector containing the genes, a strain, a preparation method, and an expression method thereof.

The invention discloses Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent and the establishing method, which belongs to the biological engineering field. The collection number of the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent is CGMCC No.1853. The invention has the advantages that the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent built by the invention can prevent the generation of excessive manna saccharify, when expressing extrinsic glucoprotein, reduce the immunogenicity of the pressed protein, and therefore, the invention has important application value in the biomedical field, and other fields; simultaneously, the strain can also be applied to the further gene knockout or the metabolic engineering reconstruction. The invention also builds a method for knocking out the alpha-1, 6-mannosyl transferase gene through secondary homologous recombination, mutant strain can be relatively easily obtained through the method, therefore the sieving workload is greatly reduced, and the success ratio is improved.

The invention relates to a gene recombination human collagen fusion peptide segment. The overall length of the peptide segment comprises 839 amino acids, the nitrogen end of the peptide segment comprises a type III human collagen peptide section of 908-1137, which totally contains 229 amino acids, and the carbon end of the peptide segment comprises a type I human collagen peptide segment of 497-1104, which total contains 608 amino acids, wherein the two peptide segments are connected by using glutamic acid and phenylalanine. The preparation method of the peptide segment comprises the following steps of: constructing cDNAs of clone type I (COL 1) and type III (COL 3) human collagen genes and a Pichia pastoris recombinant expression vector pPOC9K-COL3a1-COL1a1; constructing Pichia pastoris genetic engineering bacteria SMD1168-COL3a10COL1a1 of the gene recombination human collagen fusion peptide segment; preparing the gene recombination human collagen fusion peptide segment, and the like. Proved by tests, the gene recombination human collagen fusion peptide segment prepared by adopting the method can be used as a material for preparing cosmetics.

The invention provides a SOD gene from animal and a method for cloning and expressing the SOD gene in colibacillus and yeast cells, wherein a nucleotide sequence of superoxide dismutase is shown as SEQ NO.1; an amino acid sequence of the superoxide dismutase is shown as SEQ NO.2; carriers of nucleotide molecules are colibacillus plasmids or yeast plasmids; cells of the nucleotide molecules are formed by carrier conversion; and cells of the nucleotide molecules of the superoxide dismutase contain colibacillus containing the nucleotide molecules or converted by the carriers or pichia yeast containing the nucleotide molecules or converted by the carriers. The invention can prepare recombinant production strains which can efficiently express and secrete Cu/Zn-SOD, realizes the production industrialization of the Cu/Zn-SOD, and achieves good pH stability, favorable thermal stability and Cu/Zn-SOD products with anti-protease hydrolyzation capacity.

The invention discloses a recombinant pichia pastoris engineering bacterium and a method for using the recombinant pichia pastoris engineering bacterium as a whole-cell catalyst for synthesizing RA (rebaudioside A). The recombinant pichia pastoris engineering bacterium is obtained through conversion of pichia pastoris after linearization of an expression vector containing exogenous DNA as follows: the DNA sequence for coding sucrose synthetase Sus1 is represented by SEQ ID NO.3, and the DNA sequence for coding UDP-glycosyl transferase UGT76G1 is represented by SEQ ID NO.4. The intracellular expression of the Sus1 and the surface expression of the UGT76G1 are realized by the aid of the recombinant pichia pastoris engineering bacterium, and the recombinant pichia pastoris engineering bacterium is used as the whole-cell catalyst and can be effectively used for synthesizing the RA, so that the synthetic efficiency of the RA is improved, the raw material utilization rate is increased, the production cost is reduced, and a new industrial way is opened for production and processing of the RA.

A process is provided for the bioconversion of a carbon substrate to 1,3-propanediol by a single organism utilizing microorganisms, such as, Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas, containing the genes encoding for an active glycerol or diol dehydratase enzyme by contacting these organisms with a carbon substrate under the appropriate fermentation conditions. Specifically, Citrobacter and, Klebsiella provide the source of exogenous genes for such active dehydratase enzyme.

The invention relates to a method of cultivating biological flocs and an application thereof in breeding litopenaeus vannamei and relates to cultivation of biological flocs. The cultivated biological flocs comprise bacillus pumilus, lactobacillus acidophilus and pichia guilliermondii. The method comprises the following steps: preparing a bacillus pumilus inoculant, a lactobacillus acidophilus inoculant and pichia guilliermondii powder; adopting a way of splashing bacillus pumilus powder, saccharomycetes powder and a lactic acid bacteria inoculant for a water body per mu every day, and arranging an nano aerating tube in the lower layer around a cultivation pond to aerate to form the flocs; adjusting the C/N weight of a cultivating water body and controlling the C/N at (12-14): 1; and after the biological flocs are cultivated, splashing the bacillus pumilus inoculant, pichia guilliermondii powder and a lactobacillus acidophilus inoculant to the water body per mu every day. The cultivated biological flocs can be applied to breeding litopenaeus vannamei.

The invention discloses an engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application, and belongs to the genetic engineering field. A trypsin gene obtained through in-vitro amplification is fused with a leading short peptide YVEF, and is wholly connected to a Pichia pastoris GS115 chromosome to construct engineered Saccharomyces cerevisiae efficiently secreting and expressing recombinant trypsin, and the recombinant trypsin having an improved stability is obtained after purification, and the heat stabilities of the recombinant trypsin at 40DEG C, 50DEG C and 60DEG C are 1.77, 2.6 and 31 times wild trypsin respectively, so the problem of the low stability of trypsin is solved. The production of trypsin through applying the engineered Saccharomyces cerevisiae has the advantages of high output, simple technology, intelligible heredity and application background of engineered Saccharomyces cerevisiae, and convenient industrial application.

The invention discloses a method for producing a recombinant human type II collagen single chain by Pichia pastoris. The present invention provides a nucleotide sequence encoding the recombinant humantype II collagen single chain, the nucleotide sequence is shown as SEQ ID NO. 1, and a nucleotide sequence of the recombinant human type II collagen single chain coded by the nucleotide sequence SEQID NO. 1 is shown as SEQ ID NO.2. The recombinant human type II collagen single chain obtained according to the present invention comprises a full-length amino acid sequence of a mature human type IIcollagen alpha1 chain, which can effectively support the realization of the biological function of the type II collagen, and has good biological activity, an amino terminal and a carboxy terminal contain different affinity purification tags, which can realize effective purifying to obtain the full-length single-chain protein, and is also beneficial for the identification of collagen; and accordingto the expression characteristics of Pichia pastoris, the method systematically optimizes the gene sequence encoding the recombinant human type II collagen single chain, so that better expression inPichia pastoris is realized.

The invention discloses a method for improving pichia pastoris recombinant expression hyaluronidase, and belongs to the technical field of bioengineering. A sectional signal peptide sequence is fused at the front section of recombinant hyaluronidase gene, so that the problem of low yield of the original recombinant strain is solved, and the yield of the recombinant hyaluronidase in the pichia pastoris is obviously improved. The successful establishment of engineering lays a certain foundation for further reducing the production cost of hyaluronidase of leech and widening the application range, and the pichia pastoris recombinant expression hyaluronidase is suitable for industrial production.

The invention relates to a pichia yeast engineering strain Pichia pastoris MS18 expressing thermostable manganese superoxide dismutase (Mn-SOD) gene Mn-sod obtained from Thermoascus aurantiacus var. levisporus. The manganese superoxide dismutase gene Mn-sod, which is obtainded from Thermoascus aurantiacus var. levisporus through the RT-PCR method, is cloned and inserted into pichia yeast integration expression vectorp PIC9K, and then the resulting manganese superoxide dismutase gene Mn-sod expression vector pPIC9K/Mn-sod is induced into pichia yeast GS115. After that, a yeast engineering bacteria MS18 expressing manganese superoxide dismutase is seleceted. The engineering bacteria is induced and cultured for six days, so the enzyme expression quantity is 0.92mg/mL, the enzyme activity is 2324 U/mL, and the molecular weight is 86.0 kDa. The engineering strain has stable activity at 50 DEG C and 60 DEG C, the enzyme activity remains 50% after heat preservation at 80 DEG C for 40 min and remains 20% after heat preservation at 90 DEG C for 60 min. The engineering strain of the invention has thermal stability, which can be used for production strain of thermostable manganese superoxide dismutase and has important economic value and social value.

The invention discloses a fusion protein and a coding gene and application thereof. The fusion protein provided by the invention is formed by connecting any one of the following proteins at the carboxyl terminal of human interleukin-10 protein: 1) human IgGFc-gamma 1 or mutant protein thereof, and 2) human IgGFc-gamma 2 or mutant protein thereof, wherein the amino acid sequence of the human interleukin-10 protein is an amino acid sequence from 1st to 160th site of the amino terminal of the sequence 1 in a sequence table. Proved by experiments, the IL-10-IgG/Fc fusion protein of interleukin-10activity is produced by using a pichia pastoris fermentation expression system, and a foundation for clinically researching the treatment effect of the IL-10-IgG/Fc fusion protein in the next step.

The invention discloses a method for preparing a recombinant human papillomavirus 18-typed L1 protein with a pichia pastoris expression system and comprises the steps of loading an HPV 18 L1 gene in accordance with optimal design into an expression carrier, transforming pichia pastoris and cultivating a transformant, thus obtaining the recombinant human papillomavirus 18-typed L1 protein, which is self-assembled into virus-like particles inside a pichia pastoris body; wherein, nucleotide sequences of the HPV 18 L1 gene in accordance with optimal design are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6. The HPV 18 L1protein that is prepared by the method of the invention has high expressed quantity. The invention further discloses a method for preparing a papillomavirus 18-typed infection vaccine with better activity by utilizing the virus-like particles of the recombinant HPV 18 L1 protein.

The invention discloses an organic biology padding for efficient livestock and poultry and a production method thereof, and belongs to the filed of fertilizer. The organic biology padding for efficient livestock and poultry comprises the following parts: 65 to 70 percent of plant fiber crushing materials, 12 to 15 percent of broken rice bran, 12 to 15 percent of clinker or fly ash, 3.5 to 4.5 percent of molasses, 0.6 to 0.8 percent of urea or diammonium phosphate, 0.2 to 0.4 percent of sodium chloride or potassium chloride, and 0.2 to 0.5 percent of inoculated strain, wherein the inoculated strain is the mixture of bacillus licheniformis, bacillus subtilis and pichia membranefaciens Hansen. According to the formula, a strain is inoculated for fermentation, and then the pH value is tested until the pH value is 5.0 and the final temperature is 60 DEG C; the pH value is kept within 5.0 to 6.0; and after 10 to 15 days, the fermentation is completed. The organic biology padding for efficient livestock and poultry and the production method thereof solve the problem of severe organic fecal pollution in the livestock and poultry farming industry of our country, can sustainably utilize and convert the livestock and poultry feces as resources into biological organic fertilizer, and achieve zero emission of livestock and poultry farming thoroughly.

The invention provides reorganized mannase, genetically-engineered bacteria of the recombined mannose and a hydrolyzing preparation mannan oligosaccharide method. The hydrolyzing preparation mannan oligosaccharide method comprises the steps 1), optimizing and reorganizing beta-mannase genes according to pichia pastoris codon preference; 2), constructing expression vector pHBM905BDM-Man containing the beta-mannase genes, converting pitchia pastoris GS115 competence cells, and fostering and checking to obtain single-copy genetically-engineered bacteria; 3) constructing, fostering and checking to obtain multi-copy genetically-engineered bacterium; 4) using different-copy-number genetically-engineered bacteria to efficiently express and prepare reorganized beta-mannase; and 5) using reorganized beta-mannase hydrolysis mannan substrate to produce mannan oligosaccharide. Substrate hydrolysis concentration is 15-40%, temperature is 50 DEG C to 55 DEG C, and time is 0.5 hour to 2 hours. At present, two-copy beta-mannase genetically-engineered bacteria GS115/MAN78(CCTCCNo:2012554) is high in expression level. The activity of the beta-mannase is 5000U/mL (a decimal number system method), hydrolysis substrate specificity is high, and hydrolysis substrate concentration is obviously higher than that of other methods.