High-efficiency synthesis of human alpha-endorphin and magainin in the erythrocytes of transgenic mice: a production system for therapeutic peptides.
Chemical synthesis of peptides, though feasible, is hindered by considerations of cost, purity, and efficiency of synthesizing longer chains. Here we describe a transgenic system for producing peptides of therapeutic interest as fusion proteins at low cost and high purity. Transgenic hemoglobin expression technology using the locus control region was employed to produce fusion hemoglobins in the erythrocytes of mice. The fusion hemoglobin contains the desired peptides as an extension at the C end of human alpha-globin. A protein cleavage site is inserted between the C end of the alpha-globin chain and the N-terminal residue of the desired peptide. The peptide is recovered after cleavage of the fusion protein with enzymes that recognize this cleavage signal as their substrate. Due to the selective compartmentalization of hemoglobin in the erythrocytes, purification of the fusion hemoglobin is easy and efficient. Because of its compact and highly ordered structure, the internal sites of hemoglobin are resistant to protease digestion and the desired peptide is efficiently released and recovered. The applicability of this approach was established by producing a 16-mer alpha-endorphin peptide and a 26-mer magainin peptide in transgenic mice. Transgenic animals and their progeny expressing these fusion proteins remain health, even when the fusion protein is expressed at > 25% of the total hemoglobin in the erythrocytes. Additional applications and potential improvements of this methodology are discussed.
Sharma
A
A
DNX Biotherapeutics Inc., Princeton, NJ 08540.
Khoury-Christianson
A M
AM
White
S P
SP
Dhanjal
N K
NK
Huang
W
W
Paulhiac
C
C
Friedman
E J
EJ
Manjula
B N
BN
Kumar
R
R
D000595:Amino Acid Sequence; D000818:Animals; D004765:Enteropeptidase / Q000096:biosynthesis; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D005914:Globins / Q000096:biosynthesis; D006454:Hemoglobins / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D010452:Peptide Biosynthesis; D010455:Peptides / Q000097:blood; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000097:blood / Q000302:isolation & purification; D018822:alpha-Endorphin / Q000096:biosynthesis* / Q000097:blood / Q000302:isolation & purification
D006454:Hemoglobins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D018822:alpha-Endorphin; D005914:Globins; D004765:Enteropeptidase