Novel purification scheme and functions for a C3-binding protein from Streptococcus pneumoniae

Biochemistry
Q ChengM K Hostetter

Abstract

To isolate microbial proteins capable of binding the third component of complement (C3), we coupled the free sulfhydryl group of methylamine-inactivated C3 to a thiolSepharose matrix. This simple technique facilitated the purification of the first C3-binding protein isolated from a bacterium (Streptococcus pneumoniae). Both metastable (native) and thioester-disrupted C3 were recognized by this protein; binding of C3 was noncovalent, independent of thioester conformation, and preferential for the C3 alpha-chain. Sequencing of amino-terminal and internal peptides from the C3-binding protein disclosed a proline-rich region spanning approximately 20 amino acids and a signal peptide that had not been previously reported. The gene was isolated from a library of genomic DNA from laboratory strain CP1200 by screening with a 1200 bp PCR product amplified from degenerate oligonucleotides encoding the amino terminal sequence and the internal proline-rich sequence. The open reading frame spanned 1692 bp; all peptide sequences were identified in the translated gene product, which also contained at least three choline-binding repeats at the carboxy-terminus. The gene was conserved, and the translated protein was functionally active in pneumo...Continue Reading

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Related Concepts

Microorganism
Colostral IgA
Immune Response
Choline
Bacterial Proteins
Abnormal Degeneration
Complement C3 precursor
Thioester
Amino Acids, I.V. solution additive
Methylamine

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