TH12985Y - Plasmid vectors for the production of target proteins outside the bacterial cells Lactobacillus plantarum and Bacillus subtilis. - Google Patents

Abstract

The plasmid vector described in this invention is a usable plasmid for the extracellular production of target proteins in host bacteria that are Generally Recognized As Safe (GRAS) by the FDA (US Food and Drug Administration). These bacteria are currently used in genetic engineering for the production of extracellular target proteins in two species: Lactobacillus plantarum and Bacillus subtilis. This plasmid vector offers a new alternative for use, with a single structure providing comprehensive benefits for both of these bacterial species. Furthermore, its composition facilitates efficient extracellular production of each bacterial target protein, making it convenient and easy to use. It reduces the need for protein extraction compared to traditional protein production, thus lowering production time and costs. The plasmid vector is characterized by the presence of a signal peptide sequence related to bacterial protein extracellular transport, isolated from the bacterium L. plantarum BCC9546. Choose from the base sequences shown in Figure 1a, 1b, or 1c.

Claims (6)

1. Plasmid vectors for the production of target proteins outside the bacterial cells of Lactobacillus plantarum and Bacillus subtilis are characterized by the presence of a sequence of base sequences associated with protein synthesis outside the bacterial cell, isolated from the bacterium Lactobacillus plantarum BCC9546. The chosen sequence can be from any of the sequences shown in Figure 1a, 1b, or 1c. 2. Plasmid vectors, as per claim 1, which contain the following core sequences for extracellular protein production: a. Origin of replication (ORI) sequence and replication protein (Rep 2) sequences in isolated Gram-positive bacteria. a. The sequence of plasmid replication in Gram-negative bacteria derived from the pUC18 plasmid. c. The promoter sequence isolated from the L-lactate dehydrogenase enzyme gene of L. plantarum BCC9546, as shown in Figure 2. d. The sequence of proteins involved in bacterial exocrinement, isolated from L. plantarum BCC9546, selectable from the sequences shown in Figure 1a, 1b, or 1c. e. The sequences specific to three restriction enzymes (Multiple cloning sites, MCS): EcoRl, Kpnl, and Smal. f. The sequence of erythromycin resistance protein. 3. A plasmid vector under claim 1 or 2, where the plasmid vector contains all the base sequences selectable from any one of the base sequences in Figure 4a, 4b, or 4c. 4. A plasmid vector, according to one of claims 1-3, that is linked to the target gene. 5. Selectable host bacteria from L. plantarum or B. subtilis containing a plasmid vector, as per any one of claims 1-4. 6. The use of a host bacterium containing a plasmid vector, as per claim 5, to amplify the target gene or express the target protein gene to produce the target protein outside the cell.