Expression And Activity Analysis Of Subtilisin QK And Single-chain Insulin Analog SCI-59 In Lactic Acid Bacteria

Lactic acid bacteria(LAB),which are widely used in the food industry,are present in the intestines of most animals,including humans.Because many have been granted "generally regarded as safe"(GRAS)and probiotic status,these bacteria can be used as live vehicles for antigenic,functional proteins or for DNA delivery to the mucosal surface.With the development of the genomics and genetic tools of LAB,it has become a research hotspot that using the recombinant LAB secreting or displaying antigentic or other functional proteins as their oral delivery system.In this thesis,we employed LAB to secret or surface display Subtilisin QK and single-chain insulin(SCI)analog SCI-59,and further analyzed their bioactivity,respectively.Thrombolytic therapy has been the main route to treat thromboembolic diseases and microorganism is one of the main sources of thrombolytic agents.Purified from the supernatant of Bacillus subtilis QK02 culture broth,Subtilisin QK is a type of effective thrombolytic reagent that has great exploitable potential.However,the unbearable flavor that occurs with fermentation and the complicated methods that are required to obtain pure products limit the application of this enzyme.The secretory expression and surface display using LAB may popularize Subtilisin QK-2 more easily and conveniently with minimal adverse effect.Insulin plays an important role in drug therapies for diabetes mellitus and as the main route of insulin delivery,subcutaneous injection may cause many side effects.Therefore,oral delivery of insulin is more preferred.However,there is a low bioavailability due to insulin degradation by proteolytic enzymes and severe pH conditions along the gastrointestinal tract.In order to use the food-grade bacteria LAB as oral delivery vehicles,a new and bioactive single-chain insulin(SCI-59)analog,containing the insulin B-and A-chains connected by an eight-residue linker was designed.Using LAB secreting or displaying biologically active SCI-59 analog would open new prospects for oral hypoglycemic agents.In this thesis,QK and SCI-59 were successfully expressed in LAB.Our innovative results were presented as follows:1.Using nisin-controlled expression system(NICE),Subtilisin QK was expressed successfully in two forms using LAB.For the secretory expression in Lactococcus lactis,Subtilisin QK was efficiently secreted into the culture using the promoter PnisA and signal peptide SPUsp.The expression levels were not different in L.lactis NZ9000 and NZ3900 without the effect of different selection markers.However,leaky expression was only detected in NZ3900.The biological activity of this secreted QK was enhanced by modulating the pH of medium to slightly alkaline during induction and by codon optimization of either the entire gene sequence(qk’)or only the propeptide gene sequence(qkpro’).For surface display onto nonviable LAB bacterial-like particles(BLPs),n LysM repeats from the C-terminal region of the major autolysin AcmA of L.lactis were fused to either the C-terminus(n = 1,3,5)or the N-terminus(n = 1)of the Subtilisin QK.These fusion proteins were secreted into the culture medium,and the QK-3LysM was able to bind to the surface of various LAB GEM particles without a loss of fibrinolytic activity.Compared to the free-form Subtilisin QK,the QK-3LysM displayed on the surface of GEM particles was more stable in the simulated gastric juice.2.According to the design principle of single-chain insulin,an eight-residue linker(RSRGLPFR)was introduced into the insulin B-and A-chains and produced the codon-optimized gene sci-59.It was was secretory expressed in L.lactis NZ3900 without using an antibiotic resistance gene and displayed onto the surface of various BLPs without genetic modification.Both the free SCI-59 and SCI-59 displayed on the surface of BLPs are biologically active as assayed by their ability to stimulate Akt signaling in differentiated 3T3-L1 adipocytes.Modification of the pH of the medium by NaOH addition at early time during induction can enhance the bioactivity of SCI-59.The C-terminal fused anchoring domain,three LysM repeats,does not affect the formation of disulfide bonds and/or the folding of SCI-59,and SCI-59 could be exposed properly and fully when SCI-59-3LysM bound to the surface of BLPs.Compared to the free form SCI-59,SCI-59 displayed on the surface of BLPs is more stable in simulate gastric juice.In this thesis,QK and SCI-59 were successfully secreted or displayed by LAB,respectively.By oral administration of recombinant LAB secreting QK or SCI-59,these two valuable functional proteins can be synthsised and secreted in the gastrointestinal tract.For QK or SCI-59 displayed on the surface of LAB BLPs,BLPs can protect them from degradation when passing through the gastrointestinal tract and therefore improving their bioavailability.Therefore,it may open new prospects for possible oral treatments of thromboembolic diseases or diabetes using live LAB secreting or BLPs carrying bioactive QK or SCI analogs,respectively.