Molecular cloning, expression, purification, and characterization of shorter forms of human glutamic decarboxylase 67 in an E. coli expression system

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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Purification and Characterization of Human Pancreatic Polypeptide Expressed in E. coli

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.2122 ◽

1995 ◽

Vol 213 (1) ◽

pp. 239-248

Author(s):

Y.V. Griko ◽

M.D. Kapanadze

Keyword(s):

Pancreatic Polypeptide ◽

Purification And Characterization ◽

E Coli ◽

Human Pancreatic Polypeptide

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Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

10.17504/protocols.io.bxxxpppn ◽

2021 ◽

Author(s):

Cristina Hernandez Rollan ◽

Kristoffer Bach Falkenberg ◽

Maja Rennig ◽

Andreas Birk Bertelsen ◽

Morten Norholm

Keyword(s):

Protein Production ◽

Expression System ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

New Family ◽

Lytic Polysaccharide Monooxygenases ◽

Strain Bl21 ◽

Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

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