Abstract
The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation – a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP), remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat) pathway, but actively fold in the periplasm following general secretory pathway (Sec) and signal recognition particle (SRP) mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.
Highlights
Since its discovery in 1962 [1] and subsequent cloning of the wt- requirements for a chosen application
Green fluorescent protein in 1994 [2] the jellyfish Aequorea consensus engineering [8] approaches are facilitated by the decline in fluorescent proteins are the most widely used reporter proteins in all gene synthesis prices, which allows the introduction of silent areas of biology
The structure of correctly folded Green Fluorescent Protein (GFP) consists of an internal many new fluorescent proteins meet or exceed some properties of fluorophore surrounded by a tight beta-barrel [18]
Summary
Since its discovery in 1962 [1] and subsequent cloning of the wt- requirements for a chosen application. Green fluorescent protein in 1994 [2] the jellyfish Aequorea consensus engineering [8] approaches are facilitated by the decline in fluorescent proteins are the most widely used reporter proteins in all gene synthesis prices, which allows the introduction of silent areas of biology Due to their unique independence from cellular mutations for codon usage adaptation [6,9,10], restriction site chaperones and non proteinogenic compounds Specific properties, site directed mutagenesis approaches where applied Despite the oxygen dependent chromophore maturation process [3], more and more for fine-tuning of variants to meet the formation of active fluorescent protein is inhibited in oxidizing environments [20-23].
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