Abstract
The context sequence motif surrounding the ATG initiation codon influences mRNA translation efficiency and affects protein production; however, the optimal sequence differs among species. To determine the optimal sequence for production of recombinant proteins in a transgenic silkworm, we compared 14-nucleotide context motifs around the ATG (ATG-context) in 50 silkworm genes and found the following consensus: (A/T)AN(A/T)ATCAAAatgN. We were also able to define the least-common motif: CCN(C/G)CGN(C/T/G)(G/C/T)(T/G)atgC, which served as a negative control. To examine the regulatory role of these motifs in protein expression, we constructed reporter plasmids containing different ATG-context motifs together with either the luciferase gene or an enhanced green fluorescent protein (EGFP) gene. These constructs were then used for comparison of luciferase reporter activity and EGFP production in BmN4 cells in vitro as well as in transgenic silkworms in vivo. We detected 10-fold higher luciferase activity in BmN4 cells transfected with the consensus ATG-context motif construct, compared to the negative control plasmid. ELISA measurements of EGFP translation products with the corresponding constructs in BmN4 cells showed consistently similar results. Interestingly, the translation efficiency of the novel consensus ATG-context motif did not show the highest activity in the transgenic silkworms in vivo, except for the fat body. The highest efficiency in the middle and posterior silk glands was produced by the sericin 1 context. Our results show that the ATG-context motifs differ among silkworm tissues. This result is important for the further improvement of the transgenic silkworm system for the production of recombinant proteins.
Highlights
There is an increased demand to develop an efficient bioreactor for the production of recombinant proteins for pharmaceutical and/or diagnostic uses
The consensus motif obtained from the alignment showed that ‘A’ was the most frequent nucleotide at positions -10, -9, -7, -6, -3, -2, and -1 (A of the ATG initiation codon corresponds to +1) within the context sequences of -10 to -1 using the binominal test (Figure 1)
Effects of ATG initiation codon sequence context on reporter genes in BmN4 cells To examine the effects of the ATG-context motif, we designed a number of variants of context sequences
Summary
There is an increased demand to develop an efficient bioreactor for the production of recombinant proteins for pharmaceutical and/or diagnostic uses. The current recombinant protein production systems using transgenic silkworms utilize mainly silk glands. The sericins are produced in the middle silk gland (MSG) region, while the fibroins are secreted in the posterior part of the silk glands (PSG). Intact human collagen produced in the PSG is not transferred to the lumen, whereas there is no problem with its secretion in the MSG (Adachi et al 2010; Tomita 2011). Projects involving the production of fluorescent color proteins, spider silks, cytokine, human growth factor and mini-collagen as a fusion protein with fibroin H or L chains were performed in PSGs (Iizuka et al 2013; Teule et al 2012; Kurihara et al 2007; Tomita et al 2003). The production of recombinant proteins in transgenic silkworms has already been established, problems regarding increasing productivity and posttranslational modifications need to be addressed (Tatemastu et al 2012)
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