11results about How to "High specific activity" patented technology

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. Wild type T7 RNA polymerase is modified to obtain the T7 RNA polymerase variant, the enzyme specific activity of the T7 RNA polymerase variant is remarkably improved compared with that of the wild type T7 RNA polymerase, the addition amount of the polymerase in the in-vitro transcription reaction process can be reduced, and the cost is saved; in addition, the mutant also effectively reduces the generation of incomplete fragment impurities, and the mRNA integrity is significantly improved to 99%. The invention further provides a method for preparing RNA through in-vitro transcription.

The invention relates to the technical field of biological medicines, in particular to a preparation method of 18F-DOPA and an F-18 labeled precursor of 18F-DOPA. The structural formula of the F-18 labeled phenolic precursor compound is as shown in a formula I: # imgabs0 #; wherein R1 and R2 are hydroxyl protecting groups; r3 is a leaving group; r4 and R5 are independently hydrogen or an amino protecting group; and R6 is a carboxyl protecting group. The invention provides a simple and efficient < 18 > F-DOPA preparation method, and particularly relates to preparation of < 18 > F-DOPA by constructing a key phenol precursor compound and performing a simple and efficient photocatalytic deoxidation fluorination reaction on aromatic hydrocarbon. The method has the advantages of few steps, high yield, simplicity and convenience in operation, good repeatability and good automatic production potential. The radioactive tracer agent 18F-DOPA synthesized by using the method has high specific activity, high chemical purity and high radioactive purity, and has an important value for clinical popularization of 18F-DOPA.

The invention discloses a method for preparing < 18 > F-m-fluorobenzylguanidine, and solves the technical problems of complex reaction steps, low radiochemical yield and low specific activity in a method for synthesizing < 18 > F-mFBG. The invention comprises a phenolic ether precursor compound of a core skeleton corresponding to 18F-mFBG, a synthesis method of the phenolic ether precursor compound, and a method for realizing F-18 labeling and preparation of mFBG by using the precursor compound. Specifically, 18F-mFBG labeling and preparation are realized by constructing a key phenolic ether precursor compound and performing simple and efficient photocatalytic deoxidation fluorination reaction on aromatic hydrocarbon. The method has the advantages of few steps, high yield, simplicity and convenience in operation, good repeatability and good automatic production potential. The radioactive tracer 18F-mFBG synthesized by the method is high in specific activity, chemical purity and radioactive purity, and has important value for clinical popularization of targeted NET molecular function PET imaging.

The application discloses an S-methyl-5'-thioribose kinase mutant and application thereof in ribose-1-phosphate synthesis, relates to the technical field of genetic engineering and enzyme engineering, and specifically discloses a high-activity S-methyl-5'-thioribose kinase mutant. The mutant is obtained through directional mutation, key mutations occur at the 128th position and the 240th position, and the catalytic efficiency on ribose-1-phosphate is significantly improved. The mutant is suitable for industrial production of ribose-1-phosphate and derivatives thereof.

The invention discloses a purification method of vilasidase beta and a liquid medicine, and relates to the technical field of protein purification. The purification method of the vilasidase beta comprises the following steps: hydrophobic charge-induced chromatography treatment, anion exchange chromatography treatment, virus inactivation treatment, cation exchange chromatography treatment, hydrophobic chromatography treatment, primary ultrafiltration treatment, virus removal filtration treatment and secondary ultrafiltration treatment, so as to obtain the purified vilasidase beta. The vilasidase beta obtained by the purification method is high in recovery rate and stable in enzyme activity.

The present invention provides an industrial separation and purification method of DNaseⅠ, belonging to the field of biotechnology. The industrial separation and purification method of DNaseⅠ of the present invention comprises the steps of centrifuging a cell fermentation liquid or a cell lysate expressing a recombinant DNaseⅠ gene to obtain a supernatant, concentrating and ultrafiltration to replace the liquid, anion exchange chromatography, affinity chromatography, composite chromatography and post-treatment. The separation and purification method of the present invention has simple steps, high yield, low cost, and is easy to industrially scale up production. The purity of the recombinant DNaseⅠ obtained by separation and purification is higher than 95%, the specific activity of the enzyme is higher than 10KU / mg, the endotoxin residue is less than 1EU / mg, and there is no RNase contamination. It can be well applied to DNA removal, DNA labeling by nick translation method, DNaseⅠ footprint analysis experiment, etc. It is widely used and has good economic value and social significance.

The application discloses a two-stage high-density fermentation method for producing active nickel-iron hydrogenase. The method comprises the following steps: performing aerobic high-density fed-batch fermentation on recombinant Escherichia coli co-expressing nickel-iron hydrogenase structural genes and mature auxiliary protein genes, so that the cell density reaches OD 600 ≥40; then switching to anaerobic conditions, adding an inducer and metal ion cofactor, and inducing expression and active center assembly of the enzyme; and finally, breaking the cells in an anaerobic environment to obtain active enzymes. Through the time-decoupling strategy, the volume yield is improved to more than 900 mg / L under the premise of ensuring high specific activity (≥20 U / mg) of the enzyme, and the technical bottleneck of low cell density and limited yield in traditional anaerobic fermentation is solved, which is suitable for industrial production of nickel-iron hydrogenase and other oxygen-sensitive metal enzymes.

The invention relates to a protein glutaminase mutant, a coding gene, a recombinant vector, a recombinant bacterium, a preparation method and application, and relates to the technical field of bioengineering, the protein glutaminase mutant has a sequence with amino acid mutation as shown in SEQ ID NO: 1, the amino acid mutation site is any one mutation site of 63W, 65I, 87A, 102S, 136M, 144P, 193S, 204F, 211V, 261F, 262G, 264L, 271V and 289M or a combination of at least two mutation sites of 63W, 65I, 87A, 102S, 136M, 144P, 193S, 204F, 211V, 261F, 262G, 264L, 271V and 289M. According to the invention, active protein glutaminase is directly generated through a double-plasmid co-expression system, a single-site mutant and a combined mutant of the protein glutaminase are constructed through site-specific mutagenesis, and the specific enzyme activity and thermal stability of the provided mutants are greatly improved; the method has important significance in improving the solubility and emulsibility of protein in food and the like, and is more beneficial to meeting the requirements of social production.

The invention provides an elution buffer solution for purifying a recombinant blood coagulation factor VIII and application of the elution buffer solution. The elution buffer solution is a buffer system containing copper ions, amino acids and / or pharmaceutically acceptable salts thereof. The concentration of the copper ions is 1-400 [mu] M. The buffer solution is used for separating and purifying rFVIII from a biological sample, the HCD residue is low, the Protein A residue is low, an rFVIII intermediate product or preparation obtained by using the buffer solution is high in specific activity and purity, and an rFVIII separation and purification method developed by using the buffer solution or an rFVIII preparation preparation method developed by using the buffer solution is high in rFVIII activity yield and product yield, beneficial to capacity expansion and suitable for large-scale production.

The invention provides chymotrypsinogen and application thereof, and particularly relates to a protein tag for improving the activation efficiency of recombinant chymotrypsinogen. Compared with naturally extracted human chymotrypsinogen, the recombinant chymotrypsinogen fused with the protein tag has significantly improved activation efficiency. The invention also relates to a recombinant chymotrypsinogen containing the protein tag, a nucleic acid molecule for coding the recombinant chymotrypsinogen, a vector containing a nucleotide sequence of the nucleic acid molecule, a nucleic acid construct and a host cell. In addition, the invention also relates to a method for expressing the recombinant chymotrypsinogen.

The application provides a Bst DNA polymerase domain, a mutant and application thereof, and belongs to the technical field of molecular biology. The amino acid sequence of the Bst DNA polymerase domain is shown in SEQ ID NO:1. The Bst DNA polymerase mutant is obtained by replacing the natural DNA binding domain of the wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO:1. The Bst DNA polymerase mutant exhibits extremely high catalytic efficiency on dUTP, and also has excellent hot-start characteristics, and can reduce the false positive rate of LAMP technology to close to zero. The Bst DNA polymerase mutant also has extremely strong complex sample tolerance, and the detection sensitivity of the pathogen is 20 times higher than that of the wild-type Bst enzyme, and can meet the early and accurate diagnosis requirements of trace infection.