32 results about "Yeast chromosome" patented technology

The invention belongs to the technical field of bioengineering, and particularly relates to a production method for a yeast recombinant collagen, which includes construction of recombinant pichia pastoris engineered strain and production of constitutively and secretively expressed recombinant collagen without methanol induction. The invention first utilizes conventional codons of pichia pastoris at the genetic level to optimize a collagen gene sequence and performs artificial total gene synthesis, the optimized collagen gene sequence is then integrated into yeast chromosome, and thereby a constitutively and secretively expressed recombinant pichia pastoris engineered strain is constructed. The fermentation process is easy to operate, yield is high, and the separation and purification of the product are simple; methanol induction is not needed, the fire-proof and explosion-proof indexes of production equipment and factory buildings are low, and the production environment is environmentally friendly and pollution-free; and no toxic and harmful substances are use, so the product is safer. A specific affinity purification label is carried, the product with higher purity can be easily obtained, and the product can be applied to biomedical materials.

The invention discloses genetically engineered protease K. Enzymatic activity of the protease K is at least two times that of conspecific natural protease, production period can be shortened to 3-4 days, and production efficiency is greatly improved. The production method comprises the following steps of performing synthesis and amplification to obtain protease K genes; cloning and transforming to integrate the protease K genes to yeast artificial chromosome; selecting a high-quality bacterial strain capable of expressing target proteins effectively by screening yeast; performing high-density fermentation to express the protease K by using a high-density fermentation technology; and performing separation and purification on protein to obtain the high-purity and high-activity protease K.

The invention relates to an antimicrobial polypeptide, and a preparation method and application thereof, belonging to the field of biomedicine. The amino acid sequence of the antimicrobial polypeptide provided by the invention is disclosed as SEQ ID NO.2. The preparation method comprises the following steps: after linearizing the coding sequence of the antimicrobial polypeptide with restriction endonuclease, transforming into Pichia pastoris GS115 by an electric shock method, recombining onto the Pichia pastoris chromosome by homology, screening high-expression strains with G418, and inducingthe expression with methanol, thereby obtaining the antimicrobial polypeptide. The test proves that the antimicrobial polypeptide has the functions of inhibiting and killing multiple bacteria, viruses, tumor cells, carcinomatous solid tumors, fungi and protozoa, can not easily generate resistance, and thus, can be used as an ideal antibiotic substitute, biological medicine for human and livestock, feed additive, preservative, bactericide or the like.

The invention describes a yeast for the dietary therapy of diabetes and a construction method thereof. A saccharomyces cerevisiae genome DNA as a template is used for amplifying two sections of rDNA sequences; pYX212 as a template is used for amplifying a nutritional marker gene URA3 and an expression cassette segment driven by a triosephosphate isomerase promoter; the four amplified products are respectively cloned at corresponding loci of pUC18, and thereby an integrating vector pNK1 is obtained. A DNA synthesization point mutation technique is then utilized to remove the restriction sites of dipeptidase VI and trypsin in the natural GLP-1 molecule, so that type I primer segments, type II primer segments and left and right connected filling primer segments are synthesized, and a ten-repeat tandem rolGLP-1 sequence is obtained by the method of step-by-step annealing and one-step connection, and is cloned in the pNK1, so that pNK-GLP is obtained. The pNK-GLP is then linearized, saccharomyces cerevisiae BJ2407 is transformed, and after screening, recombinant yeast SG2 is obtained. Because the rolGLP-1 gene is integrated in yeast chromosome, the recombinant strain can stably and efficiently express the characteristics of the rolGLP-1 and reduce the blood sugar level of a rat with hyperglycaemia, and can be used in the development of yeast biomedicine for treating diabetes.

The invention relates to the field of microorganisms, in particular to a transfer method for saccharomyces cerevisiae chromosomes. A synthetic No.V chromosome saccharomyces cerevisiae strain and a synthetic No.X saccharomyces cerevisiae strain are fused into a diploid strain through an endoreduplication backcross method, the wild No.V chromosome and No.X chromosome are induced to be lost through galactose, and screening is conducted through an SC+5-FOA culture medium; a saccharomyces cerevisiae haploid strain carrying the synthetic No.V and No.X chromosomes is obtained through spore splitting. Compared with existing achievements, the transfer method has the advantages that transfer of the saccharomyces cerevisiae chromosomes is rapidly achieved; cross swap of sister chromatids is avoided, and the saccharomyces cerevisiae haploid strain with the chromosomes transferred is obtained.

The invention discloses yeast recombined human-source I-type collagen alpha1 chain protein, a synthesis method and an application thereof. The recombinant human-source I-type collagen alpha1 chain protein is composed of N-terminal affinity purification marker, human-source I-type collagen alpha1 chain mature peptide sequence and C-terminal affinity purification marker, and the double specific affinity purification markers are designed at two ends of the protein, so that purification of the recombinant protein is facilitated, and detection and full-length identification of the recombinant protein are facilitated. The synthesis method includes utilizing pichia pastoris idiomatic codon to optimize a collagen gene sequence and performing artificial whole-gene synthesis on a gene level; building a recombinant vector through PCR, enzyme digestion and connection, and integrating the recombinant vector into yeast chromosome to build a recombinant pichia pastoris engineered strain secreting andexpressing the recombined human-source I-type collagen alpha1 chain protein. The double specific affinity purification markers carried by the protein is supportive of two-step specific purification,and easiness in acquiring a high-purity product is realized.

The invention relates to the technical field of gene engineering, and particularly relates to yeast for large-scale gene rearrangement and a construction method thereof. According to the yeast for large-scale gene rearrangement and the construction method thereof, a complete yeast chromosome is cut off by using a Vika/vox technology, so that homologous recombination is generated by chromosome centromeres, the whole chromosome is knocked out, six synthetic chromosomes are integrated, and a large-scale rearrangement event is generated. Compared with existing achievements, the yeast for large-scale gene rearrangement and the construction method thereof have the following beneficial effects that cutting of the yeast chromosome is achieved simply, efficiently and quickly; cross exchange of sister dyeing monomers is avoided, and haploid yeast integrated by the six synthetic chromosomes is obtained; and very large-scale genome rearrangement is generated simply, efficiently and quickly.

The invention relates to the technical field of biology and in particular discloses a method for knocking out a saccharomyces cerevisiae chromosome. According to the method, a Vika/vox technology is utilized for removing a complete yeast chromosome, and chromosome centromeres are specifically recombined into a ring by virtue of loci, so that knockout of the whole chromosome is realized. Compared with a method for realizing elimination of the whole chromosome in a reduction mitosis manner, the method disclosed by the invention has the advantages that cutting of the saccharomyces cerevisiae chromosome is more simply, efficiently and rapidly realized, crossing over of sister chromatids is avoided, and a saccharomyces cerevisiae homozygous diploid bacterial strain with the chromosome knocked out is obtained.

A method for expressing a desmodus rotundus salivary plasminogen activator in pichia pastoris is realized by the following steps: artificially synthesizing the full-length gene of the desmodus rotundus salivary plasminogen activator (Bat-PA) in vitro, then cloning the full-length gene on an expression vector pPIC9K and then transforming a linearized recombinant plasmid to a yeast cell by electroporation to be stably integrated on a yeast chromosome. The method stably and efficiently expresses the Bat-PA, especially DSPAalpha1 in pichia pastoris, thus lowering the production cost, improving the expression quantity and ensuring the generated recombinant protein to have the bioactivity of the natural protein. The purified protein can be used for treating various thrombotic cardio-cerebro vascular diseases.

The invention discloses a pichia yeast recombinant bacterial strain highly expressing a Neuritin truncated protein, and an application thereof. The recombination bacterial strain is Pichia pastoris and has a preservation number of CGMCC NO:9912. Compared with Neuritin truncated proteins expressed by Escherichia coli and sold in the present market, the Neuritin truncated protein expressed by the pichia yeast recombinant bacterial strain has the following advantages: an exogenous protein expressed by eukaryotic cells has post-translational modification bioactivity, and exogenous gene is stably integrated in yeast chromosome, and can realize stable passage and easy large scale culture. A suitable concentration of Neuritin can promote the regeneration and function recovery of early stage sciatic nerves after injuries, provides a new method for treatment of peripheral neuropathy injury, and has good application prospect.

The invention discloses design and construction methods and application of standardized bio-elements. The construction method of the standardized bio-elements comprises the following steps that 1, a target genophore group, a promoter carrier group and a terminator carrier group are constructed. A series of standardized bio-elements are designed and constructed based on a yeast system, restriction enzymes II can be utilized for assembling the standardized bio-elements into an ultralong foreign DNA fragment, the foreign DNA fragment is a target product synthesis route composed of multiple genes, and the fragment is integrated to a specific site of a yeast chromosome through yeast homologous recombination for stable expression; a heterogenetic source in the target product yeast can be synthesized, optimum expression of the target product can be achieved by optimizing the promoters of all the genes, and therefore unique advantages and great significance are achieved in the optimization process of target product synthesis.