Microorganism expressing active d-proline reductase, and method for producing active d-proline reductase
A proline and microorganism technology, which is applied in the field of microorganisms expressing active D-proline reductase and producing active D-proline reductase, and can solve problems such as increasing demand
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Embodiment 1
[0091] Example 1: Expression of PrdA and PrdH Proteins
[0092] 1-1: Gene selection for PrdA protein expression
[0093] In order to express the PrdA protein, the amino acid sequence of the D-proline reductase PrdA protein (Q17ZY9 , UniProtKB, SEQ ID NO: 3) were tested. The nucleotide sequence of D-proline reductase PrdA protein (CD630_32440, NCBI GeneBank AM180355.1, SEQ ID NO: 4) was used to construct the expression vector.
[0094] 1-2: Construction of the vector expressing PrdA (pETDuet_H6-prdA-H6)
[0095] A 6X histidine tag (H6) for Western blot analysis was incorporated into both ends of the nucleotide sequence, and genes having NdeI and XhoI restriction enzyme sequences at the amino and carboxyl terminals, respectively, were synthesized. The synthesized gene and the pETDuet-1 vector were specifically digested with NdeI / XhoI restriction enzymes, respectively, and each gene fragment was then obtained by separation on agarose gel. A vector (pETDuet_H6-prdA-H6) expre...
Embodiment 2
[0110] Embodiment 2: Expression of PrdB protein
[0111] 2-1: Gene selection for PrdB protein expression
[0112] For PrdB protein expression, the amino acid sequence of the D-proline reductase PrdB protein (Q17ZY6, UniProtKB , SEQ ID NO:5) were tested. The nucleotide sequence of the protein (CD630_32410, NCBI GeneBank AM180355.1, SEQ ID NO: 6) was used to construct an expression vector.
[0113] 2-2: Construction of PrdB expression vector (pCDFDuet_prdB-H6)
[0114] Genes were synthesized by incorporating a 6X histidine tag (H6) at the carboxyl terminus of the nucleotide sequence for Western blot analysis and including NdeI and XhoI restriction enzyme sequences at the amino and carboxy termini, respectively. The synthesized gene and pCDFDuet-1 vector were specifically digested with NdeI / XhoI restriction enzymes, respectively, and each gene fragment was then obtained by separation on agarose gel. A vector (pCDFDuet_prdB-H6) expressing PrdB was constructed by enzymatic re...
Embodiment 3
[0133] Example 3: Functional verification of PrdH protein and evaluation of D-proline reductase activity including PrdH protein expression estimate
[0134] 3-1: Expression of D-proline reductase protein to verify PrdH function
[0135] Based on the information demonstrated above, the expression of D-proline reductase was attempted and for this purpose the following combinations of vectors were evaluated.
[0136] 1) Co-expression of PrdA and PrdB proteins
[0137] Co-express the previously constructed vector expressing PrdA protein (pETDuet_H6-prdA-H6) and the vector expressing PrdB protein (pCDFDuet_prdB-H6-P selC _selC and pACYCDuet_selA_selB vectors). These three expression vectors were transformed into Escherichia coli BL21(DE3) and cultured in LB medium (50 ml) containing antibiotics (0.1 mg / ml ampicillin, 50 μg / ml spectinomycin and 30 μg / ml chloramphenicol) Incubate (37°C, 200 rpm).
[0138] 2) Co-expression of PrdA, PrdB protein and PrdH protein
[0139] The p...
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