Effects of the 20-kilodalton helper protein on Cry1Ac production and sporeformation in Bacillus thuringiensis

Bacillus thuringiensis produces large amounts of various pesticidal protein s during the stationary phase. In order to achieve a high yield and form cr ystals, some pesticidal proteins require the presence of other proteins. He lper protein P20 is required for efficient production of both the Cyt1A and Cry11A crystal proteins in B. thuringiensis subsp. israelensis. Although f ull-length Cry1 protoxins are usually independent in terms of expression an d crystallization in B. thuringiensis, in this study P20 significantly enha nced production of Cry1Ac protoxin (133 kDa) in an acrystalliferous and pla smid-negative strain. In the presence of P20, the yield of Cry1Ac protoxin increased 2.5-fold, and on average the resulting crystals were 1.85 mum lon g and 0.85 mum wide, three times the size of the crystals formed in the con trol lacking P20. Correspondingly, the recombinant strain that coexpressed P20 and Cry1Ac exhibited higher toxicity against Heliothis armigera larvae than the control. Furthermore, serious degradation of Cry1Ac in vivo was ob served, which has seldom been reported previously. Actually, most protein w as completely degraded during synthesis, and after synthesis about one-thir d of the expressed protoxins were degraded further before crystallization. In this process, P20 protected only nascent Cry1Ac from degradation, indica ting that it acted as a molecular chaperon. In addition, spores were smalle r and rounder and had a thinner exosporium layer when they were produced in the presence of P20. In summary, Cry1Ac was severely degraded during synth esis; this degradation was effectively relieved by P20, which resulted in e nhanced production. Our results indicated that P20 is an effective tool for optimizing protein production in vivo.