Cloning, expression and purification of Ppl-1, a kappa-chain binding protein, based upon protein L from Peptostreptococcus magnus.
Protein L is a multi-domain, cell wall constituent of certain strains of Peptostreptococcus magnus, which binds to the variable domain of the L-chains of Ig. A gene fragment which codes for a single Ig-binding domain of protein L (Ppl-1) was cloned into a modified pKK223-3 vector and over-expressed in E. coli JM103. A rapid protein purification protocol is described. In these studies, purified Ppl-1 was immobilised on to an agarose gel and tested against an array of Igs and Ig fragments. It was found that Ppl-1-bound Igs from a number of different sources via interactions with the L kappa-chain. An enzyme linked immunosorbant assay has been developed to assay the binding of Ppl-1 to IgG. The incubation of Ppl-1 with human serum does not produce an immunoprecipitate, thus suggesting one unique interaction per binding domain which has been confirmed by ELISA. These experiments demonstrate the potential value of Ppl-1 as an immunological tool and as an affinity chromatography ligand for the purification of Igs.
Bottomley
S P
SP
Department of Biochemistry, University of Southampton, Bassett Crescent East, UK.
Beckingham
J A
JA
Murphy
J P
JP
Atkinson
M
M
Atkinson
T
T
Hinton
R J
RJ
Gore
M G
MG
D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000378:metabolism*; D008222:Lymphokines / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010462:Peptostreptococcus / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D029584:Prostatic Secretory Proteins
D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D008222:Lymphokines; D029584:Prostatic Secretory Proteins; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors