Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments.
Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.
Hayhurst
A
A
Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland.
Harris
W J
WJ
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001435:Bacteriophages; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000096:biosynthesis*; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C072721:Skp protein, E coli