Amplified expression and large-scale purification of protein G'.
PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.
Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK.
J P
JP
Murphy
T
T
Atkinson
R
R
Hartwell
G B
GB
Stevens
R J
RJ
Hinton
C R
CR
Goward
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli; D005798:Genes, Bacterial; D006358:Hot Temperature; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013291:Streptococcus / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins