第二部分為醱酵槽培養，分別以SSP、modified R及高密度培養基利用DO-stat醱酵策略探討重組蛋白質表現之最適條件。結果顯示重組大腸桿菌利用SSP培養基批式培養並以IPTG誘導後，菌體濃度（OD600）可達15，Tox1最大產量為0.5 g/L，單位時間產率可達50~60 mg/L/h，在modified R培養基批式培養中，菌體濃度可達53，Tox1產量為1.5 g/L，單位時間產率可達160 mg/L/h。在高密度饋料培養條件下，菌體濃度（OD600）可達180，Tox1最大產量為5.8 g/L，單位時間產率可達370 mg/L/h。
第三部分Tox1之性質及純化，發現以高密度培養並於30oC誘導之菌體，有74%之Tox1以可溶性狀態存在，且只有28%的可溶性Tox1可利用10~35%硫酸銨沉澱。Tox1在純化過程中會逐漸降解，成為74 kDa與66 kDa兩個分子量，因此無法測得可溶性Tox1之等電點。 In this study, the effects of culture conditions on the growth of recombinant E. coli BL21 (DE3) and the expression of subunit protein of Pasteurella multocida toxin 1 (Tox1) were investigated in both shaking flask and fermentor. In shaking flask, the optimal condition for the expression of Tox1 was by culturing E. coli in LB medium at 37oC with initial pH 6.17-7.87 until mid-log phase, which is about 3-4 h after inoculation. Inducer, IPTG (2.5~400 M), was then administrated and cells were cultured at 25-37oC for another 3- 7 h and the yield of Tox1 could be as high as 0.1 g/L.
Three different media, i.e. SSP, modified R and high density culture (HCDC), were used to study the optimal condition in fermentor in dissolved oxygen-stat (DO-stat) culture. It was found that cell density of 15 OD600 units and 0.5 g/L Tox1 (50-60 mg/L/h) could be obtained when recombinant E. coli was cultured in SSP medium at 37oC until mid-log phase followed by induction and culture with IPTG (100-200M) at 25oC. Higher cell density of 53 OD600 units and 1.5 g/L Tox1 (169 mg/L/h) could further be achieved by changing to modified R culture with similar culture/induction parameters. The highest yield which was cell density of 180 OD600 units and 5.8 g/L Tox1 (370 mg/L/h), was obtained when recombinant E. coli was cultured in HCDC with feeding.
In contradict to previous literature, the majority of recombinant Tox1 (74%) was expressed in soluble form inside E. coli. Only 28% of the soluble Tox1 could be purified by precipitation with 10-30% ammonium sulfate. The purified Tox1, however, was degraded into 74 kDa and 66 kDa fragments so that no exact PI could be determined.