Nanobody production can be simplified by direct secretion from Escherichia coli.

It is well known that camelids (camels and llamas) have fully functional antibodies with only a heavy chain consisting of a single variable domain and two constant domains. This single variable domain is called a "nanobody" and many nanobodies are synthesized in the cytosol of Escherichia coli, however, most of the nanobodies become inclusion bodies without tags to enhance their solubility. We generated a vector system to enable the secretary expression of nanobodies in Escherichia coli. In this system, several NBs were secreted into the culture supernatant. Since the vector contained 6xHis tag and AviTAG, biotinylation (even fluorescent-labeled) of AviTAG was achieved during cell culture, and purification of the supernatant was a step by immobilized metal ion adsorption chromatography. The procedure described in this study is believed to be as simple as regular plasmid minipreps. Therefore, many laboratories can use this method.

Repeated inversions within a pannier intron drive diversification of intraspecific colour patterns of ladybird beetles.

How genetic information is modified to generate phenotypic variation within a species is one of the central questions in evolutionary biology. Here we focus on the striking intraspecific diversity of >200 aposematic elytral (forewing) colour patterns of the multicoloured Asian ladybird beetle, Harmonia axyridis, which is regulated by a tightly linked genetic locus h. Our loss-of-function analyses, genetic association studies, de novo genome assemblies, and gene expression data reveal that the GATA transcription factor gene pannier is the major regulatory gene located at the h locus, and suggest that repeated inversions and cis-regulatory modifications at pannier led to the expansion of colour pattern variation in H. axyridis. Moreover, we show that the colour-patterning function of pannier is conserved in the seven-spotted ladybird beetle, Coccinella septempunctata, suggesting that H. axyridis' extraordinary intraspecific variation may have arisen from ancient modifications in conserved elytral colour-patterning mechanisms in ladybird beetles.

A novel fusion protein that functions as an enhanced green fluorescent protein reporter and a tetracycline-controlled transcriptional activator.

Binary expression systems are widely employed to analyze gene function in vivo using transgenic organisms. The tetracycline-off (Tet-Off) system, which is a binary expression system that uses a tetracycline-controlled transactivator protein (tTA) and its tetracycline operator sequence (tetO) binding site, was developed as a method for temporally controlling gene expression. To facilitate the use of the Tet-Off system in animal species other than the model organisms that are widely used for genetic analysis, we constructed two different fusion proteins containing enhanced green fluorescent protein (EGFP) as the reporter gene and tTA as the transactivator, in different configurations. We assessed the utility of these fusion proteins designated as tTA-EGFP and EGFP-tTA in transgenic fruit flies. We showed that, although EGFP of both fusion proteins was efficiently fluoresced, transcriptional activation occurred only by the tTA-EGFP fusion protein. Furthermore, tetracycline (Tc) and doxycycline (Dox) both effectively inactivated tTA-EGFP, repressing gene expression under tetO control in a concentration-dependent manner. Additionally, the removal of Tc or Dox from the diet can recover the transactivator activity of tTA-EGFP in a concentration- and time-dependent manner. The tTA-EGFP fusion protein will therefore be useful in the analysis of gene function in a wide range of animal species.