104 results about "Pichia ambrosiae" patented technology

The invention discloses recombinant pichia pastoris for heterogenous high level secretory expression of a rhizomucor miehei lipase. The recombinant pichia pastoris is obtained by introducing a self leader peptide gene for encoding the rhizomucor miehei lipase by employing a molecular biology technology, then optimizing a copy number of an objective gene, and constructing a vector which can be expressed in pasteur pichia pastoris. Researches show that: the introduction of the self leader peptide greatly increases the secretion amount of the objective lipase, a pichia pastoris recombinant of a lipase gene (pro-rml) containing two copies has highest enzyme activity, and the lipase Pro-RML has higher activity of hydrolysis of tri-acylglycerol.

The invention discloses preparation and application of recombinant plectasin. The method comprises designing plectasin gene according to preferred codons of Pichia pastoris, wherein possible nucleotide sequences of the plectasin gene are expressed in SEQ ID NO. 1, constructing recombinant expression vectors pPICPlectasin and recombinant genetic engineering bacteria Pichia pastoris X33pPICPlectasin (CGMCC NO. 3564), carrying out a high density fermentation process on the recombinant genetic engineering bacteria Pichia pastoris having a high expression level, wherein a total protein concentration of supernate from the high density fermentation process is 729 microgrammes per milliliter, dialyzing and freeze-drying the supernate, and orderly carrying out a gel filtration chromatography treatment and a reversed phase high performance liquid chromatogram treatment on the freeze-dried supernate to obtain high purity recombinant plectasin. The high purity recombinant plectasin is not hemolytic, has favorable PH stability, heat stability and anti-pepsin activity, and can inhibit effectively the growth of gram-positive pathogen Streptococcus pneumonia, staphylococcus aureus and staphylococcus epidermidis. Therefore, the high purity recombinant plectasin can be utilized for treating and preventing gram-positive bacterium and especially streptococcus and has potential antimicrobial drug development values.

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.

Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., α-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

The presently disclosed subject matter concerns a process for the co-production of glucosamine polymers (chitin, chitosan or any of its derivatives) and polymers containing glucose, mannose and / or galactose, by the high cell density fermentation of the yeast Pichia pastoris in a bioreactor under aerobic conditions. The process can include the use of glycerol byproduct from the biodiesel industry as carbon source. Pure glycerol, pure methanol, glycerol-rich or methanol rich mixtures may also be used as carbon sources. The P. pastoris fermentation process can be duly optimized for attaining high cell densities and high cell wall chitin content. The disclosed subject matter also concerns polymers containing glucose, mannose and / or galactose.

The invention discloses a construction method of Pichia pastoris expressed by an OCH1 defect anti-CD20 tetravalent antibody. The method comprises the following steps: knocking out the alpha-1,6-mannosyl transferase OCH1 of a strain JC308, connecting reconstructed och1 having no expression ability and a screening label to pPICZalphaA, introducing the obtained pPICZalphaA to wild Pichia pastoris JC308, carrying out homologous recombination, screening to obtain an OCH1 defect strain denoted as detaoch1, connecting the sequence of a synthesized anti-CD20 tetravalent antibody with a Pichia pastoris expression plasmid pPIC9, and introducing the obtained product to the OCH1 defect strain detaoch1 to obtain the Pichia pastoris. The Pichia pastoris alpha-1,6-mannosyl transferase (OCH1) gene is knocked out to prevent formation of high-mannose carbohydrate chains, so glycosylation difference between yeast expression proteins and human natural proteins is shortened, and the safety of medicinal proteins is guaranteed.

The invention relates to a method for improving the expression level of Pichia pastoris recombinant protein. On the basis of the normal process flow of recombinant Pichia pastoris, trace melatonin is added to greatly improve the expression of the recombinant protein. By utilizing the method, radical accumulation in yeast brought by intimidation, such as unbalanced oxygen supply, during the high-density fermentation of the recombinant saccharomycete is lowered, and the damage on the yeast brought by the radical in the high-density fermentation process is eliminated so as to improve the physiological condition of the yeast. After the melatonin is used, the expression level of the recombinant protein is improved to a large extent.