Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.     

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60–100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90–156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.