title: Amplified expression and large-scale purification of protein L'.
abstract: The gene fragment (PPL') encoding the functional unit of peptostreptococcus protein L was isolated using PCR and expressed in E. coli. As the gene fragment lacked its own promoter, the 5' PCR primer was designed to incorporate an Nde1 restriction site (CATATG) into the gene. This enabled the gene to be cloned in frame into an Nde1 restriction site immediately downstream of a trp promoter. To prevent read through, a stop codon was introduced into the 3' primer. Expression of PPL' was up to 27% total cell protein which compares favourably to the 0.1% total soluble cell protein obtained from the original clone of peptostreptococcus. Following a heat step homogeneous PPL' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in yields of 90%.
authors.affiliation: Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK.
authors.forename: J P
authors.initials: JP
authors.lastname: Murphy
authors.forename: M
authors.initials: M
authors.lastname: Atkinson
authors.forename: A R
authors.initials: AR
authors.lastname: Trowern
authors.forename: G B
authors.initials: GB
authors.lastname: Stevens
authors.forename: T
authors.initials: T
authors.lastname: Atkinson
authors.forename: C J
authors.initials: CJ
authors.lastname: Duggleby
authors.forename: R J
authors.initials: RJ
authors.lastname: Hinton
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial; D005813:Genes, Synthetic; D007074:Immunoglobulin G / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010462:Peptostreptococcus / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013728:Terminator Regions, Genetic
chemical_list: D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins