Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1994 Mar;62(3):1101-8.
doi: 10.1128/iai.62.3.1101-1108.1994.

Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli

A M Berry  1 , R A Lock, S M Thomas, D P Rajan, D Hansman, J C Paton
Affiliations

Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli

A M Berry et al. Infect Immun. 1994 Mar.

Abstract

A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert, and the putative hyaluronidase coding sequence was subcloned into pBluescript SK as a 3.8-kb PstI-ClaI fragment (designated pJCP802). The complete nucleotide sequence of this insert was determined. The region included an open reading frame sufficient to encode a polypeptide with an M(r) of 107,751. An active hyaluronidase with an M(r) of approximately 89,000 was purified to homogeneity from E. coli DH5 alpha(pJCP802). N-terminal amino acid sequence analysis of the purified protein suggested that translation initiation was occurring primarily at a TTG codon within the major open reading frame. However, immunoblot analysis using antiserum raised against the purified 89-kDa hyaluronidase indicated that E. coli DH5 alpha(pJCP802) also expressed the 107-kDa form of the enzyme. This antiserum labelled a 107-kDa protein in partially purified hyaluronidase preparations from S. pneumoniae. The hyaluronidase activity in this pneumococcal extract was also neutralized by the antiserum.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nature. 1970 Aug 15;227(5259):680-5 - PubMed
    1. Infect Immun. 1993 Aug;61(8):3234-9 - PubMed
    1. Anal Biochem. 1976 May 7;72:248-54 - PubMed
    1. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 - PubMed
    1. J Infect Dis. 1980 Dec;142(6):923-33 - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources