In addition, it is advisable to test protein orthologs of LY2157299 different origin, including distantly related or unrelated species. At this point, analysis of the primary and secondary structure of both the encoding mRNA and the translated polypeptide may anticipate downstream problems. There is a plethora of freely available software and databases for identifying protein families and sequence conservation patterns, putative signal peptides, lipoboxes, glycosylation, phosphorylation and other posttranslational modifications, transmembrane domains, and unfolded/disordered regions. Protein location within the cell, i.e. cytoplasmic, periplasmic, or extracellular, provides an indication of the requirements of the protein for proper folding, including disulfide bond formation and the need for special chaperons in each cellular compartment. Further prediction of the secondary structure content can give clues about possible protein domains and motifs, a characterisation which may prove useful for chopping full-length multi-domain proteins into globular moieties. In general, successful recombinant protein expression depends on the removal of wild-type SP, lipoboxes, posttranslational signals, low-complexity regions, hydrophobic residues at the protein termini and membrane spanning regions, while conserving the boundaries of globular domains. In parallel, cDNA characterisation is important in designing the cloning strategy and identifying potential problems at the transcriptional and translational levels. Although these processes are affected by a number of exo- and endo-nucleases, the stability of the resulting mRNA is critical in protein expression experiments. mRNA can be protected by introducing sequences at the 59 untranslated regions and stem loop structures at the 39 UTRs. The GC base content may affect levels of expression and can be easily determined by sequence analysis software. Rare codons, especially consecutive ones, are frequently found in heterologous genes and may lead to translational errors due to ribosomal stalling. Such codon bias can be remedied by replacing selected codons or, if necessary, by overall gene optimisation using appropriate software. Once the above requirements are fulfilled, the gene can be inserted into the vector by directional cloning using restriction enzymes that do not cut within the gene sequence. Efficiency of translation termination can be increased by introducing strong stop codons at the end of the translated gene. No expression system is generic for all target proteins, so both bacterial and eukaryotic systems need to be explored. Escherichia coli provides the cheapest expression host, and it is the most widely used but its machinery is not as sophisticated as that of eukaryotic hosts, and it cannot always express well folded proteins of variable origin. Other alternatives often need to be tested, including bacterial systems such as Bacillus subtilis and more advanced eukaryotic systems such as the yeasts Pichia pastoris and Saccharomyces cerevisiae, the baculovirus expression system in insect cells, mammalian cells, or cell-free systems using prokaryotic extracts, which have highly variable costefficiency ratios.