Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland.
The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.
Van Cott
K E
KE
Department of Chemical Engineering, Pharmaceutical Engineering Institute, Virginia Tech, Blacksburg 24061, USA.
Butler
S P
SP
Russell
C G
CG
Subramanian
A
A
Lubon
H
H
Gwazdauskas
F C
FC
Knight
J
J
Drohan
W N
WN
Velander
W H
WH
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D019149:Bioreactors; D019735:Carbon-Carbon Ligases / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology; D005164:Factor IX / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008894:Milk Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010314:Partial Thromboplastin Time; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D013552:Swine / Q000235:genetics*; D019076:Transgenes; D014812:Vitamin K / Q000378:metabolism
D008894:Milk Proteins; D011486:Protein C; D011993:Recombinant Fusion Proteins; D014812:Vitamin K; D018698:Glutamic Acid; D005164:Factor IX; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase