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 bacterial expression vector for secreted or surface-anchored recombinant proteins. 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 proteins for a variety of uses. In this study, the SPEX system has been further developed by the construction of vectors that express polyhistidine-tagged fusion proteins. SPEX vectors were constructed with an N-terminal or C-terminal histidine tag. The C-repeat region (CRR) from Streptococcus pyogenes M6 protein and the Staphylococcus aureus nuclease A (NucA) enzyme were tested 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-terminal position and could be purified using MAC. The M6 fusions retained immunoreactivity after expression and purification as determined by immunoblots and ELISA analyses. In addition, NucA fusions retained functional activity after MAC purification. The M6-His and NucA-His fusions were purified approximately 15- and 10-fold respectively with approximately 30% recovery of protein 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 proteins from the supernatant of recombinant S. gordonii cultures. This study further illustrates the value of the SPEX system for secreted expression and purification of proteins.
Myscofski
D M
DM
Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331-3804, USA.
Dutton
E K
EK
Bolken
T C
TC
Franke
C A
CA
Hruby
D E
DE
D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D013291:Streptococcus / Q000235:genetics*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D006639:Histidine