Feng et al. [7] expressed SK66-His and Sperstad et al. [37] expressed the GRP-denominated hystatin, that showed deleterious activity against Gram-positive and Gram-negative bacteria and yeasts. Furthermore, Shlyapnikov
et al. [36] cloned and expressed a synthetic gene in a pET vector. This gene, named Ltc2a (latarcin 2a), was fused to 6 glycine residues and further demonstrated protective activity against E. coli and Endocrinology antagonist Bacillus subtilis. The number of recombinant antimicrobial peptides expressed has greatly increased in recent years due to advances in molecular biology, allowing the establishment of strategies for expression such as host choice, promoter type and appropriate post-transcriptional modification features [33]. Bacterial systems remain highly attractive due to low cost, high productivity and rapid use. Although the bacterial systems seems to be useful for eukaryotic protein expression some limitations must be overcome, such as codon usage, incorrect folding, protein degradation by protease from host cell and host toxicity caused by heterologous protein [14]. Over-expression of heterologous proteins in E. coli often produces high quantities of insoluble protein. In order to overcome such limitations, the optimization of
expression conditions such as temperature, growth media, induction parameters, promoters and E. coli expression strain maybe used [33]. Fusion tags as Trx, NusA, GST, MBP and His6 were able to increase protein solubility, improving the purification processes by decreasing production PAK5 costs Pexidartinib in vivo and increasing yield [27] and [31]. Although fusion tags maybe interesting since they facilitate peptide solubility and purification, these tags can also often interfere within protein structure and/or function. Consequently, it is commonly recommended that the tag be removed after the purification process [27], although Carson et al. [3] have shown that the His6 tag does not normally alter the structure of recombinant proteins. Tag removal can be performed by proteolytic cleavage, but this approach can be problematic due to non-specific and inefficient cleavage or loss of protein stability and solubility [27]. Another
factor that impairs heterologous antimicrobial peptide expression in E. coli is cytotoxic AMP activity against the bacterial host. This leads to clear difficulties in scale-up and to low yields processing requires chemicals or expensive enzymes, and multistep purification of peptides reduces yield, thus making peptide production less cost effective [1]. In spite of all these problems, some studies describe the expression of AMPs in prokaryotic expression system as E. coli with yielding varying from 2 mg L−1 to 40 mg L−1 [18], [19], [33], [38] and [41]. In the present work the guava GRP gene, named Pg-AMP1, was synthesized and further expression strategy was designed for production of the recombinant peptide in a prokaryotic system.