Novel strategy for protein production using a peptide tag derived from Bacillus thuringiensis Cry4Aa

Abstract

Numerous proteins cannot be sufficiently prepared by ordinary recombinant DNA techniques because they are unstable or have deleterious effects on the host cell. One idea to prepare such proteins is to produce them as protein inclusions. Here we developed a novel system to effectively prepare proteins by using peptide tags derived from the insecticidal Cry toxin of a soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag, designated 4AaCter, facilitates the formation of protein inclusions of glutathione S‐transferase in Escherichia coli without losing the enzyme activity. Application of 4AaCter to the production of syphilis antigens TpN15, TpN17 and TpN47 from Treponema pallidum yielded excellent results, including a dramatic increase in the production level, simplification of the product purification and high reactivity with syphilis antibody. The use of 4AaCter may provide an innovational strategy for the efficient production of proteins.

In Vivo Crystallization of Three-Domain Cry Toxins

“…Hayakawa et al [14] used a peptide tag derived from the C-terminal half of the Cry4Aa protoxin (amino acids 696–851) to enhance the production and purification of functional TpN protein [14]. …”

Aggregating tags for column‐free protein purification

“…4AaCter is a polypeptide of 156 residues in length, derived from the C‐terminal region of the insecticidal Cry4Aa toxin of a soil bacterium, Bacillus thuringiensis [24, 45]. The Cry4Aa toxin normally forms protein crystals, which is mediated by its C‐terminal region, or blocks 6 and 7 of the protein [24]. The protein crystal solubilizes in the dipteran midgut alkali fluid and yields the active toxin after digestion by midgut proteases [45].…”

“…The protein crystal solubilizes in the dipteran midgut alkali fluid and yields the active toxin after digestion by midgut proteases [45]. The Sakai group found that the 4AaCter tag was able to push a number of target proteins (including glutathione S‐transferase (GST), syphilis antigens TpN15, TpN17 and TpN47) into protein aggregates upon expression in E. coli [24]. Proteins in these aggregates were likely to be active, although no measurement of activity for these protein aggregates was reported [24].…”

“…The bottleneck with the use of these tags is that refolding process is required to recover active protein [22, 23]. However, more recently, different groups have identified new tags that can induce the formation of active protein aggregates, such as ELK16, 4AaCter [10, 24, 25]. These tags eliminate the need for refolding [10, 24].…”

“…However, more recently, different groups have identified new tags that can induce the formation of active protein aggregates, such as ELK16, 4AaCter [10, 24, 25]. These tags eliminate the need for refolding [10, 24]. The third form of protein aggregates is formed in vitro upon soluble expression under certain chemical or physical conditions.…”