[Cloning and expression of non-position-specific lipase gene from Proteus vulgaris].
OBJECTIVE
To produce Proteus vulgaris lipase (PVL) in large quantities, we cloned and expressed the lipase gene in Escherichia coli.


METHODS
We cloned PVL gene by PCR method and then inserted the open reading frame of PVL gene into pET-DsbA and pMBP-P vectors. PVL gene was expressed in E. coli with the introduction of isopropyl beta-D-1-thiogalactopyranoside (IPTG). We also studied the optimal culture conditions, including the concentrations of glucose, IPTG and ampicillin, induction temperature, and pH value of the medium. The characteristics of recombinant lipase were examined after affinitive purification by His-chelating affinity chromatography.


RESULTS
The open reading frame of PVL gene consisted of 864 base pairs, encoding a protein of 287 amino acids. The sequence was deposited to GenBank with the accession number FJ643627. The gene was expressed in E. coli and active lipase was obtained from E. coli BL21 cells by the induction of IPTG, and the lipase production reached 192. 2 U/mL in BL21 [pET-PVL] after culture for 15 h at 15 degrees C. The maximum production was obtained by culturing BL21 cells in LB medium (pH 8.5) with 15 mg/mL glucose and 200 mg/L ampicillin, as well as adding 100 mg/L IPTG at OD600 of 1.2. A single protein band of 31 kDa was displayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after affinitive purification. The properties of lipase expressed in E. coli were similar to the native one, which could hydrolyze all three esters of triglyceride.


CONCLUSIONS
We have succeeded in over-expressing the lipase gene from P. vulgaris in E. coli, and this research has laid a foundation for improvement and industrial application of this lipase.
Lu
Yaping
Y
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.
Gu
Jing
J
Tang
Yanchong
Y
Lv
Fengxia
F
Bei
Xiaomei
X
Lu
Zhaoxin
Z
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011514:Proteus vulgaris / Q000201:enzymology*
D001426:Bacterial Proteins; D008049:Lipase