Loading…

Streptokinase: Cloning, Expression, and Excretion by Escherichia coli

Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage λ replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene (skc)....

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1984-06, Vol.81 (11), p.3557-3561
Main Authors: Malke, Horst, Ferretti, Joseph J.
Format: Article
Language:English
Subjects:
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage λ replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene (skc). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized. One recombinant clone was used to subclone skc in E. coli plasmid vectors. Plasmid pMF2 [10.4 kilobases (kb)] consisting of pACYC184 with a 6.4-kb H46A DNA fragment in the EcoRI site and pMF5 (6.9 kb) carrying a 2.5-kb fragment in the Pst I site of pBR322 were among the recombinant plasmids determining streptokinase production in three different E. coli host strains. Expression of skc was independent of its orientation in either vector, indicating that its own promoter was present and functional in E. coli. However, expression in pBR322 was more efficient in one orientation than in the other, suggesting that one or both of the bla gene promoters contributed to skc expression. Several lines of evidence, including proof obtained by the immunodiffusion technique, established the identity of E. coli streptokinase. Testing cell-free culture supernatant fluids, osmotic shock fluids, and sonicates of osmotically shocked cells for streptokinase activity revealed the substance to be present in all three principal locations, indicating that E. coli cells were capable of releasing substantial amounts of streptokinase into the culture medium.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.81.11.3557