Isolation of an in vivo protein-complex required for Fap1 glycosyaltion

Fap1 is a serine-rich surface protein of Strepotcoccus parasanguinis, which mediates bacterial adhesion and biofilm formation. Fap1 is a glycoprotein. Genes coding for glycosyltransferases and glycosylation-associated proteins in S. parasanguinis play important roles in Fap1 maturation and functions. The identification and isolation of glycosylation-associated protein complexes will facilitate our understanding of Fap1 glycosylation mechanism. However, current methods, like co-immunoprecipitaion and yeast two-hybrid system, can not efficiently identify glycosylation-associated proteins in Streptococci. Objective: this study is to efficiently purify glycosylation related protein complexes. Methods: A Tandem Affinity Purification (TAP) followed by mass spectrometry was used to isolate protein glycosylation complex. A putative glycosyltransferase Gtf2 that is essential for glycosylation of Fap1 was labeled with two tags: a protein G and a calmodulin binding domain at the carboxyl-terminus and constructed on a shuttle vector pVPT. The recombinant vector was introduced into the gtf2 deletion mutant to restore Gtf2 expression by the Gtf2-TAP fusion protein. The tagged Gtf2 and its associated proteins were purified by TAP method and introduced into an in vitro glycosylation assay to determine their ability to support Fap1 glycosylation. Results: The tagged Gtf2 was functional and able to support Fap1 glycosylation and bacterial adhesion. Glycosyltransferase Gtf1 was co-purified with Gtf2 by the TAP scheme. The direct interaction between Gtf1 and Gtf2 was demonstrated in in vitro GST-pull down experiment and yeast two-hybrid assay. Conclusion: The TAP method is a rapid and efficient method to purify protein complexes in Streptococci. Studies are ongoing to examine the ability of the purified protein complex to support Fap1 glycosylation. This work was supported by NIDCR/NIH.