We could apply a similar strategy to other bacterial models, we inquired whether the i-tag could likewise increase expression of fluorescent proteins in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis. As the Leucine codon also varied in the different N-terminal regions that were tested, we speculated whether the use of less frequently used codons could prevent the successful expression of the Citrine fluorescent protein, as the introduction of less frequently used codons in the beginning of mRNA molecules may result in the reduction of the translation of the encoded proteins. This hypothesis had been previously suggested for B. subtilis, where a sequence encoding the first eight aminoacids of specific ComGA was proposed to overcome the slow translation initiation caused by the eukaryotic codon bias present in fluorescent proteins. This is due to the fact that the absence of tRNA molecules can result in a stalled translation process, which consequently may lead to the disassembly of the complex ribosome/mRNA. Regucalcin was first identified in 1978 as a calcium -binding protein, which does not contain the typical EF-hand Ca2+ -binding motif. Subsequently, RGN was identified as senescence marker protein-30 based on the characteristic down-regulation of this protein with ageing in the rat liver. As the name suggests, RGN regulates intracellular Ca2+ Life Science Reagents homeostasis through the modulation of the activity of Ca2+ channels, Ca2+ -ATPase in the membrane of mitochondria and endoplasmic reticulum and – ATPase in the plasma membrane. Moreover, RGN plays an important role in the regulation of Ca2+ -dependent enzymes, such as protein kinases, tyrosine kinases, phosphatases, phosphodiesterase, nitric oxide synthase and proteases. Several studies have showed a role for RGN in the regulation of cell death and proliferation; indeed, RGN also regulates DNA synthesis and fragmentation and modulates the expression of oncogenes, tumour suppressor genes and cell cycle regulators, influencing cell survival and apoptosis. RGN has been localised to the nucleus, cytoplasm and the mitochondria. RGN is widely expressed in a variety of tissues and cell lines and was first identified in the liver, where this protein is highly expressed. However, RGN mRNA and/or protein expression has also been detected in the male and female reproductive tract, submandibular glands, several brain districts, the heart, skeletal muscle, lung, kidney, adrenal glands, bone. RGN protein has been also shown to be secreted to biological fluids, namely plasma and seminiferous tubules fluid. The expression of RGN is regulated through many factors, including intracellular Ca2+ concentration and regulatory transcription factors, namely transcription factor AP-1, b-catenin, nuclear factor I-A1 and RGN gene promoter region-related protein. In addition, Ca2+ -independent mechanisms, including hormonal factors, such as thyroid, parathyroid and sex steroid hormones, have been described in the regulation of RGN expression in cells. The regulation of RGN expression through sex steroids in the rat liver, kidney and more recently, the breast, prostate gland and testis has also been demonstrated.