377 results about "Pichia" patented technology

The invention belongs to the technical field of microorganisms, and particularly relates to a Prlisteria ataveovii strain and application thereof. The invention provides a Pichia aryabhattai B8, which is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, the preservation date is September 10, 2024, and the preservation number is CGMCC No.31932. The Pichia aryabhattai B8 disclosed by the invention has the advantages that the Pichia aryabhattai B8 can be used for preparing the Pichia aryabhattai B8; the prelisteria aryabhattai B8 disclosed by the invention has a relatively strong function of producing indoleacetic acid, and the content of indoleacetic acid in plants can be increased, so that the stress resistance of the plants is improved; dissolution of insoluble phosphorus and potassium in soil can be promoted, soil nutrients are improved, plant growth is promoted, and plant quality is improved; and meanwhile, the P.aryabhattai B8 has a certain antagonistic effect on pathogenic bacteria of the wheat root rot, and can be used for preventing and treating the wheat root rot.

The invention discloses a recombinant humanized XVII type collagen as well as an expression vector and a genetically engineered bacterium thereof. Four functional fragments with high activity are obtained through screening, one of the fragments is optimized and then combined according to a certain sequence to obtain recombinant humanized XVII type collagen JYC1703, or one of the fragments is optimized and then combined according to a certain sequence, and GPP sequences are fused at an amino terminal and a carboxyl terminal respectively to obtain recombinant humanized XVII type collagen JYC1701 and JYC1702, and the recombinant humanized XVII type collagen JYC1701 and JYC1702 are combined according to a certain sequence to obtain the recombinant humanized XVII type collagen JYC1701 and JYC1702. After codon optimization, amplifying and cloning into an expression vector, transforming into pichia pastoris competent cells after enzyme digestion linearization, and screening high-copy recombinants through G418 resistance gradient, so as to obtain pichia pastoris genetically engineered bacteria for expressing recombinant humanized XVII type collagen. The recombinant humanized XVII type collagen JYC1701, JYC1702 and JYC1703 disclosed by the invention show excellent functions of promoting cell adhesion, cell proliferation and cell migration, and are suitable for preparing products such as skin care products or medical instruments and the like.

The present disclosure relates to a recombinant collagen, peptide fragments thereof, and sequence variants thereof. A novel Pichia pastoris strain for producing the recombinant collagen, fragments thereof, and sequence variants thereof, and methods of using the recombinant collagen, fragments, and sequence variants thereof are also provided. The present disclosure fulfills a need for environmentally friendly and safe alternatives for skincare.

The invention belongs to the technical field of bioengineering, and particularly relates to recombinant III-type humanized collagen as well as a coding gene and application thereof. According to the invention, a novel III-type humanized collagen sequence is designed by adopting an advanced AI technology and molecular dynamics simulation, so that the recombinant III-type humanized collagen product is ensured to have high stability and degradation resistance. According to the invention, the screened new III-type humanized collagen sequence is transferred to the pichia pastoris strain for recombinant expression, and the prepared recombinant III-type humanized collagen has the advantages of strong stability, high expression quantity and good hydrophilicity, and meanwhile, the recombinant III-type humanized collagen also has obvious effects of promoting cell proliferation and cell adhesion.

The invention belongs to the technical field of protein engineering, and discloses a Brazilian sweet protein mutant and application thereof. According to the invention, lysine is used for replacing aspartic acid at the 29th site of Brazilian sweet protein, and / or arginine is used for replacing glutamic acid at the 36th site, and / or alanine is used for replacing aspartic acid at the 50th site, amino acid mutation at the sites is carried out to construct a Brazilian sweet protein mutant, and the Brazilian sweet protein mutant is subjected to heterologous expression in pichia pastoris GS115, so that the Brazilian sweet protein mutant is obtained. And carrying out sweetness performance evaluation on the expressed Brazilian sweet protein mutant. The sweetness property of the double mutant Asp29Lys-Asp50Ala protein is improved by 240% compared with the sweetness property of the Brazilian sweet protein before mutation.

The invention provides cat serum albumin and application thereof, and relates to the technical field of protein engineering. According to the invention, mutation is carried out on the cat serum albumin for the first time to obtain a beneficial mutation site, so that the cat serum albumin mutant which can be highly expressed in a pichia pastoris system is obtained. In addition, the cat serum albumin mutant I31L also has the effect of treating ascites due to cirrhosis, and the treatment effect is obviously higher than that in the prior art. A new biological material is provided for application of the cat serum albumin, and a foundation is laid for genetic engineering modification of the cat serum albumin.

The invention provides a recombinant XVII type collagen and application thereof, and relates to the technical field of protein engineering. The novel recombinant human XVII type collagen is obtained by optimizing and screening collagen structural domain sequences of the human XVII type collagen and then combining the sequences. The recombinant human XVII type collagen is consistent with the corresponding part of the amino acid sequence of the human XVII type collagen, does not generate immunological rejection when being applied to a human body, and can be widely applied to the industries of surgical medical treatment and medical beauty. In addition, the recombinant human XVII type collagen is high in stability, and the problem of protein degradation in the process of expressing recombinant protein by pichia pastoris can be avoided. In addition, the recombinant human XVII type collagen also has the effects of high biological activity, high biological safety, promotion of cell proliferation and differentiation and promotion of hair growth, and can be applied to the field of cosmetics or medicines as a functional component.

The invention belongs to the technical field of bioengineering, and particularly relates to recombinant III-type humanized collagen and application thereof. Area fragments with good hydrophilicity in a human III-type collagen alpha chain are selected and sequentially connected to serve as expression protein, the amino acid sequence is 100% identical to that of a corresponding part of natural human III-type collagen, and immunological rejection cannot be generated when the human III-type collagen is applied to a human body. According to the invention, the screened new III-type humanized collagen sequence is transferred into pichia pastoris for recombinant expression, the prepared recombinant III-type humanized collagen has the advantages of strong stability, high expression quantity and good hydrophilicity, and meanwhile, the recombinant III-type humanized collagen also has obvious effects of promoting cell proliferation and cell adhesion.

The invention relates to the technical field of synthetic biology, in particular to recombinant XVII type collagen as well as a preparation method and application thereof.The amino acid sequence of the recombinant XVII type collagen comprises a sequence shown in any one of SEQ ID NO.1-SEQ ID NO.3. The recombinant XVII type collagen can be highly expressed in an escherichia coli and pichia pastoris system, the expression system is simple, the cost is low, and the recombinant XVII type collagen is suitable for industrial production. The yield is high; the biological activity is good. The protein can be compounded with other proteins to form raw materials of tissue engineering products, cosmetics, health care products or medicines, so that the protein can be widely applied to the fields of medicines, medical instruments, biological materials, tissue engineering, cosmetics and the like.

The invention discloses a function-enhanced recombinant fibronectin as well as a preparation method and application thereof. The amino acid sequence of the recombinant fibronectin is shown as SEQ ID No.3, and the recombinant fibronectin is formed by fusing a main structural domain FN III8-10 formed by combining human fibronectin mutated into proline at the 171st site with integrin and a main structural domain FN III12-14 formed by combining the human fibronectin with cell factors. The recombinant fibronectin provided by the invention has enhanced cell proliferation and adhesion promoting performance of human fibroblasts, shows excellent cell biological efficacy, can be efficiently expressed in pichia pastoris engineering strains, and has wide application prospects in the fields of skin care, beauty and tissue engineering.

The invention discloses a multi-effect repair type collagen and application thereof, and belongs to the technical field of synthetic biology. Transdermal peptide TD-1, a transmembrane region of human XVII type collagen, an extracellular sixteenth non-collagen region, an extracellular fifteenth collagen region and a coding gene of a His tag are selected to be connected in series, and a gene sequence is optimized through pichia pastoris codon selection preference; and then the T-COL17R3 is constructed and expressed to obtain the T-COL17R3. Efficacy experiments prove that compared with similar products sold in the market, T-COL17R3 not only has excellent transdermal performance, but also has better cell proliferation and migration promoting capability, better capability of resisting early saccharification product ketoamine and later saccharification product dicarbonyl compounds, better capability of scavenging free radicals and resisting elastase, and can be used for preparing the anti-epidermal drug. In addition, synthesis of melanin in B16-F10 cells can be reduced, so that T-COL17R3 has excellent application potential in development of pharmaceutical compositions or skin care products with repairing, anti-wrinkle and firming or whitening and brightening effects.

The invention relates to a novel thaumatin mutant as well as a preparation method and application thereof. The invention provides a thaumatin mutant. The thaumatin mutant comprises an amino acid sequence of SEQ ID NO: 5 and a 6His tag sequence. The invention also provides a construction method of pichia pastoris engineering bacteria for expressing the thaumatin mutant and a preparation method of the thaumatin mutant. The inventor constructs a novel thaumatin mutant through genetic engineering, and large-scale expression of the thaumatin mutant is realized by using a pichia pastoris expression system. Sensory evaluation proves that the novel thaumatin mutant has high sweetness, and his-tag has little influence on the sweetness of protein. Stable isotope labeling of the novel thaumatin mutant is realized through a stable isotope labeling technology and adjustment of culture medium conditions. A 1H-15N HSQC spectrogram is utilized to prove that the his-tag basically has no influence on the protein conformation.

The invention discloses a recombinant human IV type collagen as well as a production method and application thereof. The amino acid sequence of the recombinant human IV type collagen is shown as SEQ ID No: 1. The recombinant human IV-type collagen is a truncated protein of a human IV-type collagen alpha1 chain, special expression elements and means are not needed, the yield of the recombinant human IV-type collagen can reach up to 18g / L only through extracellular secretory expression of a commercial pichia pastoris expression system, the yield is obviously higher than the high yield level of the recombinant collagen considered in the technical field, and the recombinant human IV-type collagen can be used for preparing the recombinant human IV-type collagen. The total length of the alpha 1 chain of the human IV-type collagen or other truncated proteins is obviously higher than that of the human IV-type collagen. In addition, the safety and biological activity of the recombinant human IV-type collagen are superior to the full length of the alpha 1 chain of the human IV-type collagen.

The invention discloses recombinant I-type collagen with a low-temperature self-assembly characteristic as well as a preparation method and application thereof, and relates to the technical field of genetic engineering. The recombinant I-type collagen with the low-temperature self-assembly characteristic is characterized in that an amino acid sequence of the recombinant I-type collagen is obtained by taking a human I-type collagen fragment as a basic unit and repeatedly connecting in series for n times, an amino acid sequence of the human I-type collagen fragment is shown as Seq ID NO.1, and n is an integer greater than or equal to 1. According to the invention, nucleotides of repetitive fragments of functional areas of the human-derived I-type collagen are optimized and screened, and co-expressed with hydroxyproline enzyme genes in a pichia pastoris system, so that the recombinant I-type collagen with low-temperature self-assembly characteristics is successfully obtained.

The invention relates to the technical field of gene engineering, in particular to a cat serum albumin preparation and characterization method. The cat serum albumin gene sequence is optimized, a recombinant expression vector is successfully constructed, pichia pastoris genetically engineered bacteria are prepared, high-yield expression of the cat serum albumin gene in pichia pastoris is achieved, a purification system is established and perfected, and a pure product with the purity larger than 98% is obtained through affinity exchange column purification. Compared with the natural cat serum albumin, the cat serum albumin has a secondary structure, stability and antioxidant activity which are consistent with those of the natural cat serum albumin, has the characteristic of low hemolytic activity, can realize large-scale efficient production, can be applied to development of pet foods and medicines, and has wide application value and market prospect.

The invention discloses a high-transdermal recombinant humanized XVII type collagen as well as a preparation method and application thereof, and belongs to the technical field of synthetic biology. According to the preparation method, transdermal peptide TD-1, different functional fragments of human XVII type collagen and coding genes of His tags are selected to be connected in series, gene sequences are optimized through pichia pastoris codon selection preference firstly, and then the high-transdermal recombinant humanized XVII type collagen is obtained; and then the recombinant humanized XVII type collagen T-COL17-NC16 and the recombinant humanized XVII type collagen T-COL17 are constructed by expression of the recombinant humanized XVII type collagen T-COL17. Effect experiments prove that compared with similar products sold in the market, the skin-care product has more excellent transdermal performance, can improve cell proliferation and migration ability, also has obvious ability of resisting early saccharification product ketoamine and late saccharification product dicarbonyl compounds, better ability of scavenging free radicals and resisting elastase, and can be used for preparing the skin-care product. Moreover, synthesis of melanin in B16-F10 cells can be reduced, and the compound is expected to become a novel functional biological raw material to be applied to the fields of pharmaceutical compositions or skin care products and the like.

The invention discloses a preparation method of a recombinant engineering bacterium for expressing XVII type collagen COL17A1, and relates to the technical field of gene engineering, in particular to a preparation method of a recombinant engineering bacterium for expressing XVII type collagen COL17A1, which comprises the following steps: gene synthesis and vector construction: synthesizing an optimized sequence SEQ ID No.1 of human source XVII type collagen COL17A1; adding proper restriction enzyme cutting sites at two ends of the sequence; the synthesized gene sequence is cloned to a pGAPZ A-alpha A vector, and the expression of the gene under the control of a GAP promoter is ensured. According to the preparation method of the recombinant engineering bacterium for expressing the XVII type collagen COL17A1, by using a GAP promoter, the constructed recombinant engineering bacterium can keep a stable expression level under different environmental conditions, the GAP promoter serving as a constitutive promoter can continuously and stably express recombinant protein in pichia pastoris, an inducer does not need to be added, the production process is simplified, and the production cost is reduced. The production cost is reduced, and the production efficiency is improved.

The invention relates to screening and application of a Candida antarctica lipase B mutant. Specifically, the Candida antarctica lipase B mutant provided by the invention is obtained by carrying out rational design and directed evolution combined strategy mutation on the basis of a wild Candida antarctica lipase B gene sequence, and the amino acid sequence of the mutant is shown as SEQ ID NO.3. The invention also relates to a preparation method of the Candida antarctica lipase B mutant. The mutant has higher enzyme activity and thermal stability, the hydrolytic catalytic activity of the mutant is improved to 2.1 times of the original hydrolytic catalytic activity, and the half-life period of the mutant enzyme at 50 DEG C is prolonged by 4 times. On the basis, pichia pastoris is used as a host to express the mutant, and a strain with hydrolytic activity improved by 52% is obtained through high resistance screening. The lipase is prepared into an immobilized enzyme to be used in a synthetic reaction of chlorogenic acid derivatives, the highest conversion rate of chlorogenic acid reaches 74% in 24 hours, and the immobilized enzyme has a good industrial application prospect.

The invention discloses a self-assembly enzyme for directional synthesis of uniform low molecular weight dextran, the self-assembly enzyme is named as YG-SpyTag-SpyCatcher-padex, the YG is a dextran sucrase gene (Genbank No.DQ345760), the padex is a dextran enzyme gene (Genbank No.KF999646.1), the SpyTag and the SpyCatcher are two specific oligopeptides, and aspartic acid in the SpyTag and lysine in the SpyCatcher spontaneously form an isopeptide bond. The invention belongs to the technical field of biology, and relates to an enzyme engineering technology. According to the invention, dextran sucrase derived from an escherichia coli genetically engineered bacterium BL21 (DE3)-YG-SpyTag and dextran enzyme derived from a pichia pastoris genetically engineered bacterium X33-SpyCatcher-padex are combined through a SpyTag-SpyCatcher protein covalent coupling system, and the obtained self-assembled enzyme can be used for catalyzing the substrate sucrose to be directly converted into dextran with the molecular weight of 3000 to 4000 Da.

The invention provides a Pichia megaterium BA1251 and a method for producing polyhydroxyalkanoate (PHA) by utilizing the Pichia megaterium BA1251 through semi-solid shallow tray fermentation. The Pichia megaterium BA1251 has the advantages that the Pichia megaterium BA1251 can be used for producing the polyhydroxyalkanoate (PHA) by the aid of the Pichia megaterium BA1251; the dry weight of zymophyte produced by the semi-solid fermentation method can reach 100g / kg, and the semi-solid fermentation method has the advantages of high yield, short period, low cost, less waste and environmental friendliness.

The invention belongs to the technical field of biological detection, and discloses a kit and a method for quantitatively detecting residual quantity of host cell protein of pichia pastoris in order to solve the problems of limit of quantitation and low detection sensitivity of an existing detection method. According to the present invention, the empty plasmid without the recombinant human serum albumin gene is introduced into the pichia pastoris, and purification is performed by combining the Protein A chromatographic column and the host cell protein coupling affinity column, such that the prepared polyclonal antibody can be effectively used for the quantitative detection of the residual amount of the pichia pastoris host cell protein in the biological product; the kit formed by the polyclonal antibody has good accuracy and repeatability on the quantitative detection of the host cell protein residues in the pichia pastoris source biological products, the quantification limit is 0.2 ng / mL, and the kit provided by the invention can meet the quantitative detection of the host cell protein residues in all the pichia pastoris source biological products.

The invention discloses a pichia pastoris engineering bacterium for synthesizing bakuchiol from de novo by converting methanol as well as construction and application of the pichia pastoris engineering bacterium. A heterologous p-coumaric acid synthesis pathway is introduced to neutral sites of different chromosomes of pichia pastoris, a high-yield p-coumaric acid strain is obtained through gene knockout, overexpression or heterologous expression of tyrosine and phenylalanine synthesis pathway key genes of a shikimic acid pathway, a heterologous bakuchiol biosynthesis pathway is introduced on the basis, and a high-yield p-coumaric acid strain is obtained. After overexpression of bakuchiol synthetase, endogenous overexpression or heterologous expression of MVA pathway key genes and exogenous acetyl coenzyme A supply pathway genes, methanol concentration optimization and bacterial strain His4 gene supplementation, the yield of bakuchiol is effectively increased to 91.2 mg / L and is increased by 59.8 times compared with that of an initial bacterial strain, and the yield of bakuchiol in a 15L fermentation tank reaches 692.8 mg / L. The method has the characteristics of high conversion efficiency, low production cost, convenience in preparation, wide industrial application prospect and the like.

The invention discloses a method for inducing plant disease resistance by using a hydrolase active mutant of xyloglucan specific glucanase XEG1. A pichia pastoris eukaryotic expression system is utilized to obtain the purified hydrolase activity mutant XEG1E136D protein of the phytophthora sojae core pathogenic factor xyloglucan specific glucanase XEG1, and the hydrolase activity mutant XEG1E136D protein does not have the hydrolase activity of plant cell walls and can activate the systemic resistance of plants. The XEG1E136D is overexpressed into a biocontrol bacterium trichoderma harzianum by utilizing an agrobacterium-mediated fungal transformation optimization system, and the XEG1E136D is proved to be capable of secreting XEG1E136D protein to the outside of cells and activating the broad-spectrum systemic resistance of plants. The XEG1E136D protein expressed by the trichoderma harzianum can be used as a plant vaccine to improve the prevention and treatment effect, and the trichoderma harzianum XEG1E136D protein has a wide application prospect in the field of plant disease prevention and treatment.

The invention belongs to the technical field of gene engineering, and relates to a method for efficiently integrating a target gene into a pichia pastoris genome in a multi-copy manner, which comprises the following steps: S1, constructing a pPIC9K recombinant plasmid containing a single-copy target gene or a pPICZ alpha Amut + recombinant plasmid containing a multi-copy target gene; s2, constructing and integrating a pichia pastoris strain containing the multi-copy target gene by utilizing the pPIC9K recombinant plasmid containing the single-copy target gene or the pPICZ alpha Amut + recombinant plasmid containing the multi-copy target gene; and S3, electrically transforming the recombinant plasmid constructed in the S1 into the pichia pastoris strain integrated with the multi-copy target gene constructed in the S2, and carrying out resistance screening. According to the invention, the contradiction between copy number and screening pressure in the traditional gene integration technology is broken through, and an engineering bacterium construction scheme with higher economical efficiency and stability is provided for industrial production of recombinant protein with high expression quantity.

The invention discloses an antibacterial peptide PPI45 / PPI47 as well as a preparation method and application of the antibacterial peptide PPI45 / PPI47. According to the invention, fungal defensin Plectasin is used as a template, and the self-assembled antibacterial peptide PPI45 / PPI47 is designed. The expression of the self-assembled antibacterial peptide PPI45 / PPI47 in pichia pastoris is realized by optimizing the gene sequence of the antibacterial peptide and constructing a specific expression vector, a perfect purification system is established, large-scale production can be realized, and the self-assembled antibacterial peptide PPI45 / PPI47 can be applied to the fields of antibacterial drug development, hydrogel wound treatment and the like, and has wide application value and market prospect.

The invention provides recombinant VII type collagen with a whitening effect as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. Research finds that the recombinant VII type collagen with a whitening effect, the expression quantity of the recombinant VII type collagen is improved by taking a genetic engineering technology as a means, and expression production of the recombinant VII type collagen is carried out by using a pichia eukaryotic expression system; the recombinant VII type collagen disclosed by the invention is expressed in pichia pastoris, can be efficiently secreted and expressed outside cells, and is not easy to degrade in a purification stage, so that the purification difficulty is reduced; the recombinant VII type collagen has an excellent whitening effect, is suitable for sensitive skin, does not cause skin irritation, itching and scorching hot, and has good practicability.

The invention relates to recombinant pichia pastoris for high expression of porcine pancreatic phospholipase A2 and application of the recombinant pichia pastoris, and belongs to the technical field of genetic engineering. According to the method disclosed by the invention, a vacuole sorting receptor Vps10 is knocked out by utilizing a CRISPR-Cas9 system, a CPY sorting way between a trans-golgi apparatus and a vacuole is interrupted, and ppPLA2 degradation is reduced; then, in order to relieve unfolded protein response in the endoplasmic reticulum, molecular chaperones are introduced into the genome, and synthesis of ppPLA2 is enhanced; the expression intensity of the ppPLA2 is improved by increasing the gene copy number of the ppPLA2; and finally, high-density fermentation is performed in a 3-L bioreactor, so that high-efficiency expression of the ppPLA2 in the pichia pastoris is realized.

The invention relates to the technical field of genetic engineering biology, and particularly discloses a GLP-1 analogue fusion protein as well as a preparation method and application thereof. According to the application, fGLP-1 and a mutant thereof are respectively fused with FSA, IgG4-Fc and a mutant thereof through connecting peptides to form a GLP-1 analogue fusion protein with the effects of high biological activity, long half-life period, high stability and the like, and meanwhile, after pichia pastoris codon is optimized and modified, an efficient pichia pastoris methanol-induced secretory expression system is utilized for expression; the problems of short half-life period, low biological activity, poor thermal stability, potential immunogenicity, complex production process, high purification and preparation cost and the like of the GLP-1 analogue prepared by the existing expression system and related biotechnology are solved.

The invention discloses a pichia pastoris high-secretion strain screening system and a use method thereof. According to the system, pichia pastoris is used as a host, and the core structure of the system is as follows: an AOX1 promoter, an alpha-factor signal peptide, a target protein-P2A peptide, an alpha-factor signal peptide, a Gamilus fluorescent protein-G4S connecting peptide-Gcw21 anchoring sequence-AOX1 terminator. The use method comprises the steps of vector construction, conversion of enzyme digestion recovery products, clone strain selection and activation, fermentation, inducible expression and screening, and product collection to finally obtain the strain with high yield of target protein. Through integration of bicistronic design, P2A peptide self-cleavage and cell surface display technologies, efficient screening of target protein high-secretion type strains is realized.

The invention discloses a recombinant VII type collagen as well as a preparation method and application thereof. The recombinant VII type collagen is obtained by repeating a short amino acid sequence GLPGAKGEKGAP for a plurality of times. The secretion yield of the recombinant VII type collagen in a commercial pichia pastoris expression system can be up to about 17.3 g / L, the extracellular secreted protein is basically not degraded in the fermentation process, the purity can reach 90% or above through simple ultrafiltration and decoloration, and industrial mass production can be achieved; and the recombinant VII type collagen has better biological activities of promoting autophagy, improving an endoplasmic reticulum stress state, promoting collagen secretion and inhibiting matrix metalloproteinase from degrading collagen, and shows good tightening and anti-wrinkle effects.