Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis.
A 1.6 kb cDNA fragment encoding the mature part of the human tissue-type plasminogen activator (t-PA) was subcloned into a Bacillus subtilis dual plasmid expression system [Le Grice et al., Gene 55 (1987) 95-103]. Expression of the tPA gene in this vector was regulated by the inducible Escherichia coli lac elements, as well as a strong phage-T5-derived promoter and ribosome-binding site preceding the polylinker. The 5' end of the tPA gene corresponding to the N terminus of mature t-PA was fused in phase to the third codon present in the polylinker region of the expression vector, p602/22, to form p602-t-PA. B. subtilis containing p602-t-PA, when induced with isopropyl-beta-D-thiogalactopyranoside, produced large amounts of immunoreactive t-PA (approx. 20 micrograms/ml). As expected, t-PA was not secreted into the culture media, but was localized in intracellular inclusion bodies and was found to be enzymatically inactive. However, enzymatic activity could be regained following complete reduction followed by slow oxidation of the solubilized inclusion bodies. The recombinant t-PA (rt-PA) showed, after purification, a smaller molecular size than melanoma t-PA, probably due to lack of glycosylation in the Bacillus system. Like melanoma t-PA, rt-PA exhibited tremendous stimulation of plasminogen activation in the presence of fibrin. Our results illustrate that B. subtilis, when supplied with the proper transcriptional/translational regulatory elements, can be an effective system for expression of heterologous gene products.
Wang
L F
LF
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Hum
W T
WT
Kalyan
N K
NK
Lee
S G
SG
Hung
P P
PP
Doi
R H
RH
D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D010959:Tissue Plasminogen Activator