Expression and Purification of Recombinant Hemoglobin in Escherichia coli

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis

Light scattering of rectangular slot antennas: parallel magnetic vector vs perpendicular electric vector

We study light scattering off rectangular slot nano antennas on a metal film varying incident polarization and incident angle, to examine which field vector of light is more important: electric vector perpendicular to, versus magnetic vector parallel to the long axis of the rectangle. While vector Babinet's principle would prefer magnetic field along the long axis for optimizing slot antenna function, convention and intuition most often refer to the electric field perpendicular to it. Here, we demonstrate experimentally that in accordance with vector Babinet's principle, the incident magnetic vector parallel to the long axis is the dominant component, with the perpendicular incident electric field making a small contribution of the factor of 1/broken vertical bar epsilon broken vertical bar, the reciprocal of the absolute value of the dielectric constant of the metal, owing to the non-perfectness of metals at optical frequencies

Simultaneous measurement of nanoscale electric and magnetic optical fields

Control of light–matter interactions at the nanoscale has advanced fields such as quantum optics1, photovoltaics2 and telecommunications3. These advances are driven by an improved understanding of the nanoscale behaviour of light, enabled by direct observations of the local electric fields near photonic nanostructures4, 5, 6. With the advent of metamaterials that respond to the magnetic component of light7, 8, schemes have been developed to measure the nanoscale magnetic field9, 10, 11, 12. However, these structures interact not only with the magnetic field, but also with the electric field of light. Here, we demonstrate the essential simultaneous detection of both electric and magnetic fields with subwavelength resolution. By explaining our measurements through reciprocal considerations, we create a route towards designing probes sensitive to specific desired combinations of electric and magnetic field components. Simultaneous access to nanoscale electric and magnetic fields will pave the way for new designs of optical nanostructures and metamaterials