Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins.
We have designed and constructed a DNA sequence encoding human tissue plasminogen activator (tPA) with convenient restriction sites that flank each of the domains of the heavy chain. To accomplish this, the first 1095 bases of the gene coding for the mature protein were synthesized with unique restriction sites engineered into the interdomainal regions. This synthetic construction was then ligated to a cDNA fragment of the tPA gene that encoded the active site, thus generating a full-length tPA gene. The gene products produced by Chinese hamster ovary (CHO) cells transfected with either the tPA cassette gene or the tPA cDNA gene were then compared with the tPA produced by Bowes melanoma cells to determine whether or not synthetic interdomainal amino acid changes had an effect on the biochemical characteristics of the molecule. Specifically, molecular weight, specific activity, enhancement by fibrinogen fragments and kinetic constants were analysed. None of the properties examined were significantly different from those of the native melanoma tPA. Therefore, the cassette gene described herein should provide considerable versatility and precision in the construction of tPA mutants by facilitating the manipulation of the finger, growth factor and kringle domains, and likewise should be useful in assessing the function of these domains within the tPA molecule. We present this cassette gene system as a model for the analysis of protein domain function applicable to other multi-domain proteins.
Rehberg
E F
EF
Molecular Biology Research, Upjohn Company, Kalamazoo, MI 49001.
Theriault
N Y
NY
Carter
J B
JB
Palermo
D P
DP
Hubert
E V
EV
Bergum
P W
PW
Wunderlich
C J
CJ
Erickson
L A
LA
Marotti
K R
KR
D000595:Amino Acid Sequence; D004261:DNA Replication; D005813:Genes, Synthetic; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011506:Proteins; D010959:Tissue Plasminogen Activator / Q000235:genetics*
D011506:Proteins; D010959:Tissue Plasminogen Activator