Expression and purification of histidine-tagged proteins from the gram-positive Streptococcus gordonii SPEX system

Streptococcus gordonii (S. gordonii) has been used as a gram-positive bacte rial expression vector for secreted or surface-anchored recombinant protein s. Fusion of the gram-positive bacterial N-terminal signal sequence to the target protein is all that is required for efficient export. This system is termed SPEX for Surface Protein EXpression and has been used to express pr oteins for a variety of uses. In this study, the SPEX system has been furth er developed by the construction of vectors that express polyhistidine-tagg ed fusion proteins. SPEX vectors were constructed with an N-terminal or C-t erminal histidine tag. The C-repeat region (CRR) from Streptococcus pyogene s Me protein and the Staphylococcus aureus nuclease A (NucA) enzyme were te sted for expression. The fusion proteins were purified using metal affinity chromatography (MAC). Results show that the fusion proteins were expressed and secreted from S. gordonii with the His tag at either the N- or C-termi nal position and could be purified using MAC. The M6 fusions retained immun oreactivity after expression and purification as determined by immunoblots and ELISA analyses. In addition, NucA fusions retained functional activity after MAC purification. The MG-His and NucA-His fusions were purified appro ximately 15- and 10-fold respectively with approximately 30% recovery of pr otein using MAC. This study shows that the polyhistidine tag in either the N- or C-terminal position is a viable way to purify secreted heterologous p roteins from the supernatant of recombinant S. gordonii cultures. This stud y further illustrates the value of the SPEX system for secreted expression and purification of proteins. (C) 2000 Academic Press.