81results about How to "Increased expression of soluble" patented technology

The present invention relates to a polypeptide construct comprising at least one CDR3 region, wherein at least one of the at least CDR3 regions comprises at least one substitution in the amino acid sequence YYDDHY (SEQ ID NO.1) and wherein the at least one substitution comprises: in the first position of SEQ ID NO.1 a substitution from Y to H; in the second position of SEQ ID NO. 1 a substitution from Y to S, from Y to N, from Y to F or from Y to H; in third position of SEQ ID NO. 1 a substitution from D to N or from D to E; in the forth position of SEQ ID NO. 1 a substitution from D to Q, from D to A, from D to V, from D to E or from D to G; in the fifth position of SEQ ID NO. 1 a substitution from H to Q, from H to P, from H to Y, from H to R or from H to N; or in the sixth position a substitution from Y to N.

The present invention is drawn to a method for enhancing secretional efficiency of a heterologous protein using a secretional enhancer consisting of a modified signal sequence which comprises the N-region of a signal sequence and/or a hydrophobic fragment of the said signal sequence comprising the said N-region and/or the hydrophilic polypeptide. The method of the present invention can be used not only for production of recombinant heterologous proteins by inhibiting insoluble precipitation and enhancing secretional efficiency of the recombinant protein into the periplasm or the extracellular fluid and but also for transduction of therapeutic proteins by enhancing membrane-permeability of the recombinant protein using a strong secretional enhancer.

The present invention provides an infectious bursal disease virus (IBDV) subunit antigen-containing vaccine composition, which contains an immune amount of recombinant VP2 protein and an adjuvant. According to the present invention, with the subunit gene engineering vaccine composition, the infectious bursal disease can be effectively prevented and controlled; and with the purification technology adopted by the recombinant antigen protein, the endotoxin content in the vaccine composition is significantly reduced, the side reaction of the chicken individuals is significantly reduced, and the safety of the vaccine is substantially increased.

The invention discloses a method for producing isoprene by utilizing blue algae, and the method comprises the following steps of: (1) expressing an isoprene synthetase coding gene derived from blue gum and an isoprene synthetase coding gene with an optimized codon in the blue algae; (2) applying the promotor of a cpc operon to express a mediate terpenoid synthetase coding gene contained in the blue algae; (3) enhancing the expression of an prenyl pyrophosphate isomerase coding gene; (4) expressing a fusion protein which contains a prenyl pyrophosphate isomerase and an isoprene synthetase; and (5) applying an SMUO tag to express a heterologous protein in the blue algae. The method disclosed by the invention can be used for carrying out genetic modification on the blue algae and expressing the isoprene synthetase (IspS) of a plant source in the blue algae, outstandingly increases the output of isoprene produced through the gene engineering blue algae through multiple methods, greatly widens the application range of the gene engineering blue algae by applying research results to express other heterologous proteins in the gene engineering blue algae and has wide industrial application prospect.

The invention relates to the technical field of genetic engineering, and particularly relates to a monooxygenase mutant and a preparation method and application thereof. The monooxygenase mutant is provided with any one of amino acid sequences as shown in formulas (I) and (II), wherein the amino acid sequence in the formula (I) reaches 80% consistency with the amino acid sequence as shown in SEQ ID NO.1; the amino acid sequence in formula (II) is obtained by modifying, substituting, decreasing or increasing one or some amino acid to 23 to 508 amino acid sites of the amino acid sequence as shown in SEQ ID NO.1; 1-34 amino acid is substituted; the mutant has the activity of monooxygenase. According to the method, a plurality of different sites, namely, 23-508 amino acid sites, are mutated; and the result shows that the mutants are capable of improving the activity, stability, soluble expression and selectivity of monooxygenase as well as decreasing the dosage of monooxygenase.

The invention provides tyrosine phenol lyase engineering bacteria, a construction method and applications thereof, wherein the engineering bacteria are identified as Escherichia coli, are named Escherichia coli MBP-TPL, are preserved in the China Center for Type Culture Collection on April 21, 2017, and have the preservation number of CCTCC NO: M 2017202, wherein the preservation address is Wuhan University, Wuhan, China. According to the present invention, the expression plasmid is constructed by integrally linking the tyrosine phenol lyase gene and the maltose binding protein gene so as to obtain the Escherichia coli MBP-TPL, wherein the expression plasmid can express the maltose binding protein and the tyrosine phenol lyase in the fused manner; and the activity of the fermentation unit bacterial amount of the tyrosine phenol lyase is high, and the soluble expression amount is high.

The invention discloses a glycosyltransferase mutant and a method for catalytically synthesizing rebaudioside A by using the same. The amino acid sequence of original glycosyltransferase is SEQ ID NO:1. According to the experiment, Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160 and Thr319 are screened on the basis of prediction obtained by starting from surface residues Q, N and T ofUGT76G1 and combining data analysis results of solvent accessible surface area, B-factor and the like, and finally, a UGT-SuSy system of the better mutant strain 76G1_Q72E-N196D-T319E is screened outby virtue of single-point or multi-point iterative mutation in Thr81, so as to realize efficient catalytic synthesis of the rebaudioside A. The glycosyltransferase UGT76G1 or a mutant gene thereof isconnected with a sucrose synthase gene to obtain a recombinant plasmid, and a double-enzyme co-expressed recombinant strain is constructed. By constructing a double-enzyme system, regeneration of in-vivo UDPG is realized, the problem of sources of expensive glycosyl donors is effectively solved, the cost is reduced, and application of biotechnology industry is promoted. The mutant is simple to prepare, the rebaudioside A is synthesized through efficient catalysis in a short time, and the biological enzyme catalysis method is green, environmentally friendly and pollution-free and is more suitable for current green industrial processing and production.

The invention relates to the technical field of genetic engineering, in particular to modified keto reductase mutant and a preparation method and application thereof. The modified keto reductase mutant has any one of amino acid sequences shown in (I) and (II), to be specific, the amino acid sequence (I) is least 80% the same as that shown in SEQ ID NO. 1, and the amino acid sequence (II) is formedby subjecting amino acids 12 to 214 in the amino acid sequence of SEQ ID NO. 1 to modification, substitution, deletion or addition of one or more amid acids; the substitution refers to substitution of 1-14 amino acids, wherein the modified keto reductase mutant is active the same as keto reductase. By designing mutations for the multiple sites of amino acids 12 to 214, these mutations provide improved activity, stability, soluble expression and selectivity for keto reductase and also enables less keto reductase to be used.

The invention discloses a preparation method of soluble recombinant proteins. According to the preparation method of the invention, a to-be-researched target protein and thioredoxin and His tag are expressed in a fusion manner, by optimizing an expression condition, the solubility expression amount of the target protein can be significantly improved, by utilizing the restriction enzyme cutting site characteristics of enterokinase, the purification of the target protein is rapidly realized by virtue of two-step affinity chromatography, and the purity can reach 95 percent or more. Compared withthe traditional method, by adopting the preparation method of the invention, the expression amount of the soluble recombinant protein can be significantly improved, the instability of the after-treatment renaturation and activity of an inclusion body protein can be avoided, the obtained recombinant protein is high in biological activity and relatively stable, and the application prospect for the mass production of the high-activity recombinant protein is good.

The invention discloses a foreign protein soluble expression plasmid, a preparation method thereof and application method thereof, which belong to the technical field of bioengineering. The plasmid is pET30E, wherein the DNA sequence of the plasmid is presented by Seq ID No.1. An amplified product obtained by performing PCR amplification on a plasmid template and a primer group is connected with a pMD18-t vector; an Escherichia coli clone is obtained by transforming Escherichia coli DH5 alpha; and a product obtained by performing enzyme cutting connection on the Escherichia coli clone serving as a substrate is used for transforming the Escherichia coli DH5 alpha to form soluble expression plasmid molecules. The application method comprises the following steps that: the obtained expression plasmid molecules are used to transform Escherichia coli BL21 (DE3) competent cells through thermal activation to form a recombinant Escherichia coli colony; and after a bacteroid cell is obtained through inoculation culture, the soluble expression of the expression plasmid molecule is improved. The plasmid of the invention has promotion effect on assistance of the soluble expression of foreign proteins in Escherichia coli and has a great significance for further analysis of the functions of the proteins.

The invention discloses a soluble expression increased bacillus pumilus W3 Cota laccase composite mutant, and belongs to the field of biological engineering. The composite L386W/G417L/G57F(WLF)CotA laccase gene which is constructed at the early stage of the lab and has greatly improved catalyzing efficiency (ABTS is used as substrate) is used as a template to further mutate Asp on the 501 site of the composite mutant into Gly and Lys on the 317 site into Asn. The bacillus pumilus W3 CotA laccase mutant has a soluble expression which is 3.04 times that of WT-CotA and 3.63 times that of WLF, has a catalyzing efficiency whichis 4.6 times that of WT-CotA and 1.03 times that of WLF, and has excellent pH value and temperature stability. The CotA laccase mutant is more applicable to industrial application, and has a wide application prospect.

The invention discloses recombinant Escherichia coli and a method for biosynthesizing diosmetin by using the recombinant Escherichia coli, the recombinant Escherichia coli is obtained by introducing a recombinant plasmid into host Escherichia coli, and the recombinant plasmid is constructed by connecting an encoding gene of AnFNSI after codon optimization and an expression vector pET-28a (+); The AnFNSI is flavone synthase 1 derived from angelica archang lica, the amino acid sequence of the AnFNSI is as shown in SEQ ID NO.1, and the amino acid sequence of the AnFNSI after codon optimization is as shown in SEQ ID NO.4. The recombinant Escherichia coli is used as a whole-cell catalyst to catalyze a substrate hesperetin to biosynthesize diosmetin. The method is green and efficient, does not need to use a large amount of organic solvents or toxic chemical reagents in the reaction process, and is low in cost, high in safety and easy for large-scale production.

The invention provides a gene for encoding Cap protein of porcine circovirus 2, a recombinant expression vector containing the gene and a recombinant bacterium containing the recombinant vector. The gene expresses easily in escherichia coli and comprises a sequence shown in SEQ ID NO.1. The invention further provides a method for inducing the recombinant bacterium to produce the Cap protein of the porcine circovirus 2 and application in the aspect of utilizing the protein to produce a vaccine of the porcine circovirus 2. Through the adoption of the gene for encoding Cap protein of the porcine circovirus 2 and the application thereof, the full-length Cap protein can efficiently express in escherichia coli, the operation is simple, the cost is cost, and the large-scale production is easy to achieve; the recombinant bacterium and the vaccine have wide application prospects in preventing and controlling diseases of the porcine circovirus 2.

The invention discloses a recombinant expression vector for improving soluble expression of pathogenesis-related proteins in Mongolia astragaloside. The recombinant expression vector is a recombinantpET30a-skp-AmPR-10 constructed by using pET30a as a vector and modifying AmPR-10 by utilizing an escherichia coli molecular chaperone skp. According to the invention, by a method of replacing the vector and carrying out molecular chaperone modification, the soluble expression of target proteins is improved so as to fulfill the aims of saving cost and improving yield; the exogenously expressed AmPR-10 is further subjected to activity measurement so as to show that the exogenously expressed proteins also have the nuclease activity and provide reference strategies for in-vitro expression of otherhomologous related proteins; and the activity may have the important significance for researching the RNA virus resistance ability of radix astragali.

The invention relates to a method for expressing a cascade antibacterial peptide gene by recombination and fusion. The multi-copy antibacterial peptide gene is expressed through the fusion with thioredoxin TrxA, thereby not only making use of the multiple copies to improve the proportion of a target polypeptide in an expression product, but also improving the expression amount of the cascade antibacterial peptide gene and promoting soluble expression through fusing protein, thereby overcoming the defects of low polypeptide proportion in the fusion protein by using a fusion expression single-copy antibacterial peptide gene method, and low expression amount and low solubility by using a direct cascade target polypeptide gene method. The method can realize the efficient expression of the cascade antibacterial peptide gene. Under optimal conditions, the total expression amount of the recombinant and fused antibacterial peptide exceeds 2mg/mL (extracting solution).

The invention discloses a method for obviously improving soluble expression quantity of linoleate isomerase in recombinant escherichia coli, and belongs to the field of genetic engineering. Accordingto the method, recombinant escherichia coli E.coli BL21(DE3)(pET-22b(+)/pai) is used as a starting strain; through co-expression of molecular chaperone proteins and assistance of the linoleate isomerase folding, soluble expression level of the linoleate isomerase is improved, so that enzyme activity of the linoleate isomerase is improved. Finally, a molecular chaperone system GroEL-GroES is preferably obtained, so that the enzyme activity of the linoleate isomerase is improved by 56 percent.

The invention provides a method for improving soluble expression of microbial transglutaminase (MTG) in escherichia coli. According to the method, two plasmids, pET-MTG and pA-GESP are co-transformed into escherichia coli DE3, and microbial transglutaminase is solubly expressed in an induced supernantant. The pET system is utilized for efficiently expressing MTG, and also a molecular chaperone co-expression system is established, so that the solubility of a MTG expression product is improved, and results show that the method helps to improve the soluble expression of MTG in escherichia coli with obvious expression effect, and establishes a good base for industrialized production in future.

The invention provides a recombinant protein for detecting tri-methylated modification of a histone locus and application thereof. An amino acid sequence of the recombinant protein comprises an N-terminal GST fusion protein sequence and a C-terminal Tudor structural domain sequence which are connected in series. The recombinant protein provided by the invention has high affinity to H3K4 and H4K20, can be used for replacing a commercialized antibody to perform Western Blot experiment, is high in sensitivity, has no cross linking; the production cost is remarkably reduced, the batch quality is extremely stable, and the batch production can be realized quickly.

This invention relates to the manufacture and application of the expression regulating and encoding region gene of Escherichia coli thioredoxin (TrxA). The gene is manufactured by: cloning the expression regulating and encoding region of TrxA by PCR technique that has the basic group sequence in SEQ ID No.1, and cloning it the the upstream of pET-28a promoter to promote the soluble expression of heterologous protein in Escherichia coli. The results show that TrxA and heterologous protein can combine to coexpress in non-fusion mode in Escherichia coli BL21 (DE3), and can promote the solubility of heterologous protein.