Production of recombinant human bile salt stimulated lipase and its variant in Pichia pastoris.
hBSSL and its truncated variant hBSSL-C cDNA clones were expressed in Pichia pastoris using two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45-50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized in P. pastoris and was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.
Sahasrabudhe
A V
AV
Astra Research Centre India, 18th Cross, Malleswaram, Bangalore, 560003, India.
Solapure
S M
SM
Khurana
R
R
Suryanarayan
V
V
Ravishankar
S
S
deSousa
S M
SM
Das
G
G
D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned; D005285:Fermentation; D018628:Gene Dosage; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D017077:Culture Media, Conditioned; D007527:Isoenzymes; D037102:Lectins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase