TY - JOUR
T1 - Cloning and protein expression of the sn-1(3) regioselective lipase from Cordyceps militaris
AU - Park, Jung Ha
AU - Park, Kyung Min
AU - Chang, Yoonjee
AU - Park, Jun Young
AU - Han, Jaejoon
AU - Chang, Pahn Shick
N1 - Funding Information:
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) ( NRF-2017R1A2B4009230 and NRF-2018R1C1B5045470 ).
PY - 2018/12
Y1 - 2018/12
N2 - In this study, the gene of a novel lipase with sn-1(3) regioselectivity (i.e., sn-1 or sn-3 specific) from Cordyceps militaris was successfully expressed by a heterologous expression system. Total RNA was extracted from C. militaris and then single-stranded cDNA was synthesized. The resulting C. militaris lipase (CML) gene was inserted in Escherichia coli expression plasmids [pET-29b(+), pET-26b, and pColdIII] to construct plasmids encoding CML, which were then transformed to E. coli strains BL21 (DE3), C43 (DE), C41 (DE3), and Origami (DE3) for protein expression. Although the recombinant CML expression level was high, it was overproduced in the form of inclusion bodies. Under a specific condition, the soluble form of the recombinant CML was detected using Western blot analysis; however, no enzyme activity was observed. To overcome the lack of post-translational modifications in recombinant CML, a baculovirus-insect expression system was introduced for eukaryotic lipase expression. pDualBac was used as the transfer vector, and the CML gene was fused under the control of the polyhedrin promoter. After generating the recombinant baculovirus, the active form of CML was successfully produced and its kinetic parameters were determined.
AB - In this study, the gene of a novel lipase with sn-1(3) regioselectivity (i.e., sn-1 or sn-3 specific) from Cordyceps militaris was successfully expressed by a heterologous expression system. Total RNA was extracted from C. militaris and then single-stranded cDNA was synthesized. The resulting C. militaris lipase (CML) gene was inserted in Escherichia coli expression plasmids [pET-29b(+), pET-26b, and pColdIII] to construct plasmids encoding CML, which were then transformed to E. coli strains BL21 (DE3), C43 (DE), C41 (DE3), and Origami (DE3) for protein expression. Although the recombinant CML expression level was high, it was overproduced in the form of inclusion bodies. Under a specific condition, the soluble form of the recombinant CML was detected using Western blot analysis; however, no enzyme activity was observed. To overcome the lack of post-translational modifications in recombinant CML, a baculovirus-insect expression system was introduced for eukaryotic lipase expression. pDualBac was used as the transfer vector, and the CML gene was fused under the control of the polyhedrin promoter. After generating the recombinant baculovirus, the active form of CML was successfully produced and its kinetic parameters were determined.
KW - Baculovirus
KW - Cordyceps militaris
KW - Positional specificity
KW - Protein expression
KW - sn-1(3) regioselective lipase
UR - http://www.scopus.com/inward/record.url?scp=85054362234&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85054362234&partnerID=8YFLogxK
U2 - 10.1016/j.enzmictec.2018.08.008
DO - 10.1016/j.enzmictec.2018.08.008
M3 - Article
C2 - 30243384
AN - SCOPUS:85054362234
VL - 119
SP - 30
EP - 36
JO - Enzyme and Microbial Technology
JF - Enzyme and Microbial Technology
SN - 0141-0229
ER -