Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2

AbstractStarting from total pancreatic mRNAs, the classical guinea pig pancreatic lipase was cloned using rapid amplification of 3' and 5' cDNA ends. Internal oligonucleotide primers were designed from a partial cDNA clone including the region coding for the lid domain. Using this strategy, we did not amplify the cDNA corresponding to the pancreatic lipase related protein 2 in which the lid domain is deleted. Amino acid sequences of the classical guinea pig pancreatic lipase and the related protein 2 were compared based on the primary and tertiary structures of the classical human pancreatic lipase. Their distinct physiological roles are discussed in the light of functional amino acid differences

Thiol ester role in correct folding and conformation of human α2-macroglobulin Properties of recombinant C949S variant

AbstractTo determine the role of the thiol ester in the folding of human α2-macroglobulin (α2M) in the active conformation, we have characterized a recombinant variant of α2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S α2M behaves like methylamine-treated plasma α2M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric α2M does not require a pre-formed thiol ester. Active α2M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S α2M followed by thiol ester formation and a conformational change that gives the native active state

CLONING AND EXPRESSION OF INDUSTRIALLY IMPORTANT FUNGALLIPASES

Triglyceride lipases from various organisms have a numberof potential industrial applications exemplified by their use in flavour enhancement, in production of esters and specialty fats, and in household detergents. We have cloned and expressed two fungallipases: the 1,3-positional specific lipase from Rhizomucor miehei (1,2), commercialized as Lipozyme™, and lipase from Humicola lanuginosa commercialized for use in household detergents (Lipolase™). Production of these lipases and other enzymesforindustrial application has necessitated the developmentof an efficient recombinant expression system. We have focusedour efforts on the development of Aspergillus as a recombinant host system for this purpose. Aspergillus has the capacity to secrete large amounts of active hydrolytic enzymes such as for example glucoamylases and amylases. Cloning of strong promoters from the corresponding genes (3,4) and development of a transformation- and selection system (4) has allowed expression from heterologous cDNA genesin Aspergillus oryzae. As first attempt to obtain heterologous expression we transferred an aspartic proteinase cDNA (5) from Rhizomucor miehei into A. oryzae and obtained high levels of secreted, active and correctly processed enzyme (4). These experiments have later been extended to fungal triglyceride lipases (6,7)