Expression and purification of a truncated recombinant streptococcal protein G.
The gene for Protein G from Streptococcus strain G148 was cloned and expressed in Escherichia coli. The regions on the gene corresponding to the albumin-binding domains and the Fab-binding region were then deleted by site-directed mutagenesis. The translation of regions corresponding to the cell-wall- and membrane-binding domains was prevented by introduction of stop codons upstream of these domains. This recombinant DNA sequence codes for a protein (G') that contains repetitive regions and that binds only the Fc portion of IgG, analogously to Protein A. Translation of the sequence produces a protein with an Mr of about 20,000. The nucleotide sequence differs from those published previously [Guss, Eliasson, Olsson, Uhlén, Frej, Jornvall, Flock & Lindberg (1986) EMBO J. 5, 1567-1575; Olsson, Eliasson, Guss, Nilsson, Hellman, Lindberg & Uhlén (1987) Eur. J. Biochem. 168, 319-324]. The protein can be substantially purified on a large scale by chromatography on IgG-Sepharose 4B. Homogeneous Protein G' can be prepared by anion-exchange f.p.l.c. on Mono Q HR. This Protein G' has a pI of 4.19 and SDS/PAGE gives an apparent anomalous Mr of 35,000.
Goward
C R
CR
Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Salisbury, Wilts, U.K.
Murphy
J P
JP
Atkinson
T
T
Barstow
D A
DA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D012709:Serum Albumin / Q000378:metabolism; D013291:Streptococcus / Q000032:analysis* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C034284:IgG Fc-binding protein, Streptococcus; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D012709:Serum Albumin