Production Of Recombinant Human Bile Salt-stimulated Lipase By Bovine Mammary Gland Bioreactor

Bile salt-stimulated lipase(BSSL)is a lipase expressed mainly in the exocrine pancreas and lactating mammary glands.BSSL has broad substrate specificities and capacity to hydrolyze glycerides,cholesteryl esters,fat-soluble vitamin esters,phospholipids,galactolipids and ceramides.Milk-derived BSSL contributes significantly to efficient digestion and absorption of milk fat and can compensate for the deficiency of pancreatic lipases for newborn infants especially preterm infants.BSSL is neither present in bovine milk nor in milk formula,while human milk BSSL is also inactivated after pasteurization.Therefore,additional BSSL supplement is beneficial to absorption of milk fat,especially for preterm infants who are not able to have sufficient fresh breast milk.BSSL can also be used to improve other fat mal-absorption conditions owing to the pancreatic insufficiency.Thus,BSSL is of high importance in research and clinical applications.In this study,we constructed a highly efficient human BSSL expressing vector,and then verified the expressing efficiency using a mouse model.We finally obtained transgenic cattle expressing recombinant human BSSL(rhBSSL)specifically in the mammary gland.We have successfully constructed the rhBSSL expressing vector pBAC-hLF-hBSSL-Neo.hLF-BAC(human lactoferrin gene containing bacterial artificial chromosome)sequence were selected as regulatory elements,which have previously been verified to be able to drive high efficient expression of foreign proteins.The vector includes 91 kb 5' regulatory region of lactoferrin gene,31 kb 3'regulatory region,9.8 kb hBSSL genomic DNA and a Neo gene.Transgenic mice were generated to verify the expressing efficiency of the recombinant BAC vector.Results showed that rhBSSL was specifically expressed in mouse mammary gland.The highest expression level was 843.6 ?g/ml,and the corresponding activity was 1023272.2 mU/mL.Further experiments showed that,rhBSSL could promote the use of milk fat and reduce the fat content in feces of pups.With bovine fibroblasts transfected using the recombinant BAC vector and susequent somatic cell nuclear transfer,we successfully obtained one transgenic cow with rhBSSL highly expressed in the mammary gland.The expression level of rhBSSL in colostrum was about 8.89 mg/ml and 5.79 mg/ml in normal milk,respectively,both of which were significantly higher than that of the previously reported transgenic sheep(3 mg/ml).We further established a method to purify rhBSSL from the transgenic milk using heparin gel chromatography and gel filtration chromatography.The purity of purified rhBSSL was 96.7%as shown by high performance liquid chromatography(HPLC).Physiological and biochemical tests showed that the purified rhBSSL had similar activity and isoelectric point to the natural human BSSL and both have the same N-terminal amino acid sequences.There were also no significant differences in bile salt dependence,pH stability,thermal stability and protease stability between the rhBSSL and the natural human BSSL.In conclusion,we have successfully constructed an expressing vector pBAC-hLF-hBSSL-Neo and obtained healthy transgenic cows with rhBSSL highly expressed in the mammary gland.The recombinant BSSL shows similar physiological and biochemical characteristics to the natural human BSSL.This study thus laid a good foundation for future commericial application of rhBSSL.