Expression in Escherichia coli of a cytochrome P450 enzyme with a cobalt protoporphyrin IX prosthetic group.

Unlike many hemoproteins, the prosthetic heme group of most cytochrome P450 enzymes cannot be extracted and replaced by modified heme groups. Here, we describe a procedure for generating a cytochrome P450 enzyme (CYP119) with cobalt protoporphyrin IX as its prosthetic group. This is achieved by expressing the protein in Escherichia coli in iron-limited medium and adding cobalt to the medium at the moment that inducible protein expression is initiated.

The C-terminus of p63 contains multiple regulatory elements with different functions.

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation.

The better tag remains unseen.

One of the most crucial steps in protein structure determination by nuclear magnetic resonance (NMR) spectroscopy is the preparation of highly concentrated and well behaving protein samples. Here we present a system of modular tags which allows for high level expression, sophisticated purification of full-length protein, and solubility enhancement while keeping the amount of additional resonances low. This system consists of two different expression constructs and utilizes the tight binding of human calmodulin (hCaM) to the calmodulin binding peptide (CBP), which has already been used as a purification tag.

Methyl groups as probes for proteins and complexes in in-cell NMR experiments.

Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [(13)C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.