Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli.
The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused to gpIII of PhiM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII, a stop codon between K2S and the gpIII gene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-beta-D-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/microg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.
Manosroi
J
J
Pharmaceutical Cosmetic Raw Materials and Natural Products Research and Development Center, Institute for Science and Technology Research and Development, Chiang Mai University, 50200 Chiang Mai, Thailand. pmpti006@chiangmai.ac.th
Tayapiwatana
C
C
Götz
F
F
Werner
R G
RG
Manosroi
A
A
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D018082:Kringles / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins; D010959:Tissue Plasminogen Activator