Secretion of functional human interleukin-3 from Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis is well-known for its huge capacity to produce secreted bacterial enzymes. Nevertheless, the secretion of pharmaceutically interesting recombinant proteins by this organism is frequently inefficient. This paper documents for the first time on the optimisation of B. subtilis for the production of human interleukin-3 (hIL-3), a four-helix bundle cytokine, which stimulates the proliferation and differentiation of a broad range of blood cells. By developing a host-vector system on the basis of the multiple protease-deficient B. subtilis strain WB700 and a multicopy plasmid containing two tandemly positioned strong promoters plus an efficient signal sequence, the hIL-3 protein was efficiently produced and secreted into the growth medium. As verified by SDS-PAGE, mass spectrometry and cross-linking experiments with a thiol-specific reagent, intact and properly folded hIL-3 was purified from the B. subtilis growth medium. Bioactivity tests showed that the isolated hIL-3 was able to specifically induce proliferation of the hIL-3-dependent leukaemia cell line MO7e. Using the eight-fold protease-deficient strain WB800 the hIL-3 accumulation in the growth medium was increased to levels up to 100 mg l(-1).

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor.

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.

Toward sustainable production of protein-rich foods: appraisal of eight crops for Western Europe. Part I. Analysis of the primary links of the production chain.

Increased production of plant protein is required to support the production of protein-rich foods that can replace meat in the human diet to reduce the strain that intensive animal husbandry poses to the environment. The suitability of lupin (Lupinus spp.), pea (Pisum sativum), quinoa (Chenopodium quinoa Willd.), triticale (x Triticosecale), lucerne (Medicago sativa), grasses (Lolium and Festuca spp.), rapeseed/canola (Brassica napus), and potato (Solanum tuberosum) for protein production in Western Europe was studied on the basis of a chain approach. The aspects considered are the familiarity of farmers with the cultivation of the crop, prospects for rapid crop improvement, protein production (kg/ha), protein quality (absence of unwanted substances) and familiarity with the usage for human food in Western Europe. Pea, lucerne, and grasses are the most promising, fair prospects are foreseen for lupin, triticale, rapeseed, and potato, whereas the possibilities for quinoa are judged to lag far behind. Estimated protein production for pea, lucerne, and grasses is 1250, 2500, and 2500 kg/ha, respectively.

Towards sustainable production of protein-rich foods: appraisal of eight crops for Western Europe. PART II: Analysis of the technological aspects of the production chain.

Increased production of plant protein is required to support the production of protein-rich foods which can replace meat in the human diet to reduce the strain that intensive animal husbandry poses on the environment. The suitability of lupin (Lupinus spp.), pea (Pisum sativum), quinoa (Chenopodium quinoa Willd.), triticale (x Triticosecale), lucerne (Medicago sativa), grasses (Lolium and Festuca spp.), rapeseed/canola (Brassica napus) and potato (Solanum tuberosum) for protein production in Western Europe was studied on the basis of a chain-approach. The technological aspects, which are considered in this paper, are the processing methods, and the functional and nutritional properties of the derived protein products. The overall evaluation of the technological prospects of the eight crops as a protein source for Western Europe leads to the conclusion that this part of the production chain is not decisive for that choice. Pea and lupin have a slight advantage over the other crops, because their concentrates and isolates are already commercially available.