7 results about "Maltose-binding protein" patented technology

The invention discloses an escherichia coli engineering bacterium for producing 5-ALA by an anaerobic fermentation method as well as a construction method and a fermentation method of the escherichia coli engineering bacterium. The engineering bacterium is constructed by taking escherichia coli (E. coli) ZX11 as a host and performing heterologous expression on 5-ALA synthetase (HemA) from rhodobacter capsulatus. In order to improve the soluble expression level of the HemA, the HemA is subjected to fusion expression with a maltose binding protein (MBP) tag. Furthermore, the engineering bacterium can also heterologously express a visual purple protein (PR) fused with the MBP tag. The construction method of the engineering bacteria comprises the following steps: sequentially cloning the MBP-HemA fusion gene and the PR gene to an expression vector and transferring into host bacteria. By utilizing the engineering bacteria provided by the invention, anaerobic fermentation can be carried out in a culture medium taking glucose as a carbon source, and 5-ALA can be efficiently synthesized. The invention provides an effective way for realizing green and energy-saving anaerobic biological manufacturing of 5-ALA.

This invention discloses a recombinant 2-O-sulfonyltransferase mutant and its preparation method, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (E28~N356) mutant of hamster-derived 2-O-sulfonyltransferase (L119V-R188E-A240I) with an N-terminal maltose-binding protein (MBP) tag. The recombinant 2-O-sulfonyltransferase mutant exhibits higher activity, 1.9 times that of the recombinant wild type, and greater stability, retaining 86.6% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant 2-O-sulfonyltransferase is increased by 5 times. This mutant, as a novel biocatalyst with high efficiency, stability, and controllable production costs, has significant application value and broad industrialization prospects in the industrial enzymatic synthesis of glycosaminoglycans such as heparin and heparan sulfate.

The invention relates to a method for improving the prokaryotic expression quantity of CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats) protein. Specifically, the invention relates to a method for improving the expression level of dCas9 protein in a prokaryotic cell, and a leucine zipper gene sequence and a dCas9 gene sequence are connected and introduced into the prokaryotic cell. According to the method, the high-purity and high-concentration dCas9 protein can be obtained, the expression quantity of the dCas9 protein is remarkably higher than that of a common method using maltose binding protein (MBP) as a fusion molecular chaperone, the obtained dCas9-lzip fusion protein still has the capability of being normally combined with DNA in a targeted manner under the guidance of sgRNA, and the function of dCas9 is not influenced by connection of a lzip sequence.

This invention discloses a recombinant glucuronide C5 epimerase mutant, expression vector, recombinant expression engineered bacteria, and their culture and induction methods, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (N29–N617) mutation (V173R–V197K–A239V–N269E) with an N-terminal maltose-binding protein (MBP) tag to a human glucuronide C5 epimerase (C5-epi). The recombinant glucuronide C5 epimerase mutant exhibits higher activity, 2.1 times that of the recombinant wild-type, and greater stability, retaining 72.5% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant glucuronide C5 epimerase is increased by 4-fold.

The application relates to a soluble expression vector of a steroid C14 alpha hydroxylase and a construction method and application thereof, and belongs to the technical field of biotechnology. A nucleic acid sequence coding a steroid C14 alpha hydroxylase and a nucleic acid sequence coding a soluble label maltose binding protein are expressed in a vector, the nucleic acid sequence coding the steroid C14 alpha hydroxylase is shown as SEQ ID NO. 1, and the amino acid sequence of the steroid C14 alpha hydroxylase is shown as SEQ ID NO. 2. The application establishes a soluble expression vector and a method of a fungus-derived enzyme in Escherichia coli, and provides a new idea for obtaining solubility of other CYP enzymes in the heterologous expression of Escherichia coli. The application significantly improves the electron transfer efficiency of the hydroxylation process by constructing an intramolecular hydrogen delivery system, and partially solves the problem of low overall catalytic rate caused by low electron transfer efficiency in the catalytic reaction of P450 monooxygenase.

The invention provides a method for in-vitro expression and purification of recombinant human interleukin 15 in mammalian cells, which comprises the following steps: a, providing a first expression vector comprising nucleic acid encoding a fusion protein of maltose binding protein (MBP) and recombinant human IL-15, the fusion protein comprises the following elements from the 5'end to the 3 'end: 5'-MBP-Furin restriction enzyme cutting site-His tag-enterokinase restriction enzyme cutting site (EK)-rhIL-15-3 '; b, providing a second expression vector, wherein the second expression vector comprises nucleic acid for coding furin; c, co-transfecting the first expression vector and the second expression vector to mammalian cells, and performing fermentation culture to enable the cells to express recombinant human IL-15 protein; d, centrifuging to obtain fermentation supernate from the step c; e, carrying out affinity chromatography through a His tag, so as to obtain a His-EK-rhIL-15 fragment; f, the fragment is subjected to enzyme digestion with enterokinase, affinity purification is conducted again through the His tag, a flow-through substance is collected, and the flow-through substance comprises rhIL-15; and g, purifying the flow-through substance from the step f by anion exchange chromatography to obtain the purified rhIL-15 protein.

The present application relates to heparinase fusion protein preparation technical field, specifically to the Bacteroides heparinase I fusion protein and its coding gene and preparation method. The Bacteroides heparinase I fusion protein includes heparinase I and maltose binding protein, and a connecting peptide is connected between heparinase I and maltose binding protein, and the nucleotide sequence of the connecting peptide is shown in SEQ ID NO.11 or SEQ ID NO.12. The present application can solve the technical problem of limited enzyme activity of heparinase obtained by using bioengineering technology, and has important theoretical and practical significance for establishing a high-efficiency and high-enzyme-activity heparinase recombinant expression system.