The natural gene was not expressed in this experiment. However, from prior studies, the natural gene has been shown to be poorly expressed in E. coli.
The recoded gene produced an NMR sample of 0.5-0.7 mM concentration, in contrast to wild-type protein that is soluble only up to 0.05 mM.
Catalyzes the first regulated step of protein synthesis initiation, promoting the binding of the initiator tRNA to 40S ribosomal subunits in humans
To improve solubility for NMR spectroscopy
Ito and Wagner
Optimization of codons was based on previous reports of E. coli codo usage, as follows: GCT for Ala,
CGT for Arg, AAC for Asn, GAC for Asp, TGC for Cys, CAG for Gln, GAA for Glu, GGT for Gly, CAG for
His, ATC for Ile, CTG for Leu, AAA for Lys, ATG for Met, TTC for Phe, CCG for Pro, TCT for Ser, ACC
for Thr, TGG for Trp, and GTT for Val.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Using codon optimization, chaperone co-expression, and rational mutagenesis for production and NMR assignments of human eIF2 alpha
Producing a well behaved sample at high concentration is one of the main hurdles when starting a new project on an interesting protein. Especially when one attempts to overexpress a eukaryotic protein in bacteria, some difficulties are encountered, such as low expression level, low solubility, or even lack of folded structure. Overexpression in prokaryotic systems is highly desirable for cost-effective production of different isotope-labeled samples needed for NMR studies. Here we describe generally applicable methods for obtaining highly concentrated protein samples efficiently. This approach was developed as we tried to produce a NMR-suitable sample of the 35 kDa human translation initiation factor eIF2 alpha, a protein that expresses poorly in E. coli and has very low solubility. First, an E. coli codon-optimized gene was synthesized on a thermal cycler, which increased the expression level by a factor of two. Second, we used co-expression of bacterial chaperone proteins, which largely increased the fraction of correctly folded protein found in the soluble phase. Third, we used rational mutagenesis guided by both the sequence alignment among homologues and the homology of one domain to a known fold for improving solubility and stability of the target protein by tenfold. Combining all these methods made it possible to produce from a one-liter preparation a 0.5 mM sample of human eIF2 alpha that showed well-resolved NMR spectra and enabled nearly complete assignment of the protein. These methods may be generally useful for studies of other eukaryotic proteins that are otherwise difficult to express and exhibit poor solubility.
J Biomol NMR. 28(4): 357-67.
The goal of this experiment was to optimize the eIF2alpha protein of humans for NMR spectroscopy, so as to better determine its structure. When optimized for expression in E. coli, the protein was expressed at a much higher concentration, allowing NMR spectroscopy studies to be performed on it. The gene was optimized using data from past studies. The exact codon replacements are explained in the section of Materials and Methods labeled "Synthesis of codon-optimized gene".