Isolation and characterization of metalloproteases with a novel domain structure by construction and screening of metagenomic libraries.

ABSTRACT: Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per microg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong prot

SUBMITTER: Waschkowitz T 

PROVIDER: S-EPMC2675231 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature