51. Recombinant Production of Honeybee Silks

Unlike Bombyx mori or spider silks that consist of large repetitive sequences and β-sheets, honeybees secrete four different types of small coiled-coil proteins in nearly equal proportions with a molecular weight of about 30 kDa [08Shi, 10Wei]. These proteins are non-repetitive and are rich in alanine residues, and the proteins were found to be stable in water [08Shi]. Due to their lower molecular weights and unique structure, it was supposed that honeybee silks could easily produce recombinant proteins. To examine this, four proteins (ABS 1–4 with 315, 289, 317, and 321 residues) from the Asiatic honeybee were expressed in Escherichia coli, and the structures of the proteins were studied. The yield of proteins in E. coli was 30, 30, 10, and 60 mg/mL for ABS-1–4, respectively. Corresponding molecular weights obtained for the proteins were 55, 32, 38, and 50 kDa, respectively. Proteins generated were found to have about 65 % coiled-coil sequences but with low (9–27 %) α-helix content and high% (45–56 %) of random coils. In addition, about 26–35 % of β-sheets were also discovered [08Shi]. Some of the properties of the recombinant honeybee silk proteins expressed in E. coli are listed in Table 51.1. Overall, it is seen that the recombinant production of honeybee silks was unable to generate the secondary and tertiary structure seen in native honeybee silk [10Wei]. In the native honeybee silks, the four isolated proteins are found as a complex but have weak interactions between them. In solution, α-sheets, β-sheets, and random coils coexist depending on the pH of the solution. The presence of high amounts of alanine that provides limited hydrophobic interactions was suggested to be the reason for the inability of the silks to maintain higher levels of α- or β-helices [08Shi]. In a similar study, recombinant proteins with yields between 0.5 and 2.5 g/L were obtained using honeybees (Apis mellifera), and the proteins were formed into fibers [10Wei]. Four of the distinct honeybee proteins were expressed in E. coli (Rosetta 2 DE3), and proteins were collected. Proteins containing all four components were concentrated to get the required viscosity, and the solution was manually drawn into fibers between the prongs of tweezers. Fibers were coagulated in 90 % methanol and 10 % water bath and drawn to about 2× the length and air-dried. Tensile properties of the fibers obtained are listed in Table 51.2.