Recombinant Protein Expression: Prokaryotic Hosts and Cell-Free Systems, 2021
Antibodies are globally important macromolecules, used for research, diagnostics, and as therapeu... more Antibodies are globally important macromolecules, used for research, diagnostics, and as therapeutics. The common mammalian antibody immunoglobulin G (IgG) is a complex glycosylated macromolecule, composed of two heavy chains and two light chains held together by multiple disulfide bonds. For this reason, IgG and related antibody fragments are usually produced through secretion from mammalian cell lines, such as Chinese Hamster Ovary cells. However, there is growing interest in production of antibodies in prokaryotic systems due to the potential for rapid and cheap production in a highly genetically manipulable system. Research on oxidative protein folding in prokaryotes has enabled engineering of Escherichia coli strains capable of producing IgG and other disulfide bonded proteins in the cytoplasm, known as SHuffle. In this protocol, we provide a review of research on prokaryotic antibody production, guidelines on cloning of antibody expression constructs, conditions for an initial expression and purification experiment, and parameters which may be optimized for increased purification yields. Last, we discuss the limitations of prokaryotic antibody production, and highlight potential future avenues for research on antibody expression and folding.
Publisher Summary The photosynthetic membrane systems of some purple bacteria are readily cleaved... more Publisher Summary The photosynthetic membrane systems of some purple bacteria are readily cleaved by both nonionic and ionic detergents to yield distinct subchromatophore fragments, which are characterized by the unique forms of bacteriochlorophyll contained in the particles. The types of subchromatophore fragments obtained depend largely upon the nature of the detergent, the presence or absence of carotenoids in the membrane, and the conditions used for growing the bacteria. In the bacteria examined to date, at least two fragments are produced—one containing the reaction center bacteriochlorophyll (BChl) and varying amounts of other associated BChl, while the other one containing only light-harvesting BChl. In the ease of Chromatium , fragmentation with sodium dodecyl sulfate (SDS) produces three fragments, but two of the three are quite similar in their general properties. In vivo , the BChl α (the form of BChl contained in the bacteria under consideration) exhibits an absorption band in the orange and a series of bands in the near infrared region. Although the exact locations of the absorption maxima of the three BChl a forms differ slightly in different bacteria, they are termed “B800,” “B850,” and “B890.” It is generally held that the different absorption properties of the BChl molecules in the near infrared region reflect different complexes of BChl with lipoproteins.
Recombinant Protein Expression: Prokaryotic Hosts and Cell-Free Systems, 2021
Antibodies are globally important macromolecules, used for research, diagnostics, and as therapeu... more Antibodies are globally important macromolecules, used for research, diagnostics, and as therapeutics. The common mammalian antibody immunoglobulin G (IgG) is a complex glycosylated macromolecule, composed of two heavy chains and two light chains held together by multiple disulfide bonds. For this reason, IgG and related antibody fragments are usually produced through secretion from mammalian cell lines, such as Chinese Hamster Ovary cells. However, there is growing interest in production of antibodies in prokaryotic systems due to the potential for rapid and cheap production in a highly genetically manipulable system. Research on oxidative protein folding in prokaryotes has enabled engineering of Escherichia coli strains capable of producing IgG and other disulfide bonded proteins in the cytoplasm, known as SHuffle. In this protocol, we provide a review of research on prokaryotic antibody production, guidelines on cloning of antibody expression constructs, conditions for an initial expression and purification experiment, and parameters which may be optimized for increased purification yields. Last, we discuss the limitations of prokaryotic antibody production, and highlight potential future avenues for research on antibody expression and folding.
Publisher Summary The photosynthetic membrane systems of some purple bacteria are readily cleaved... more Publisher Summary The photosynthetic membrane systems of some purple bacteria are readily cleaved by both nonionic and ionic detergents to yield distinct subchromatophore fragments, which are characterized by the unique forms of bacteriochlorophyll contained in the particles. The types of subchromatophore fragments obtained depend largely upon the nature of the detergent, the presence or absence of carotenoids in the membrane, and the conditions used for growing the bacteria. In the bacteria examined to date, at least two fragments are produced—one containing the reaction center bacteriochlorophyll (BChl) and varying amounts of other associated BChl, while the other one containing only light-harvesting BChl. In the ease of Chromatium , fragmentation with sodium dodecyl sulfate (SDS) produces three fragments, but two of the three are quite similar in their general properties. In vivo , the BChl α (the form of BChl contained in the bacteria under consideration) exhibits an absorption band in the orange and a series of bands in the near infrared region. Although the exact locations of the absorption maxima of the three BChl a forms differ slightly in different bacteria, they are termed “B800,” “B850,” and “B890.” It is generally held that the different absorption properties of the BChl molecules in the near infrared region reflect different complexes of BChl with lipoproteins.
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