Expression And Charaterization Of Bovine Pancreatic Deoxyribonuclease Ⅰ In Pichia Pastoris

Bovine pancreatic deoxyribonuclease I was first crystallizated by Kuntiz from Bos Taurus pancreas in1950, which plays an important role in the history of protein chemistry and enzymology. DNase I is thoroughly researched now, which belongs to deoxyribonuclease I. It is reported that it involved in many important physiological processes, such as salvage pathway of nucleotides, cell apoptosis, degradation of DNA, and the assembly of actin.In traditional way, Bp-DNase I is obtained through extracting directly from the bovine pancreas or expressing in the E. coli. The former method requires more steps and complex operation, which is time-consuming, and DNase I is easy to be deactived in the process of the extraction, thus it is not suitable for large-scale production. The other method cannot express Bp-DNase I efficiently because the induction time couldn’t be too long, once this enzyme accumulated to a certain concentration in the host cell, DNA will be degraded; besides, the growth of the host cell is restricted. Although it is reported that this enzyme can also be expressed in COS-1cells, but this method is merely suitable for laboratory research, and COS-1cell culture needs strict aseptic operation and costs high, which is not a good method of producing Bp-DNase I in large scale. Therefore, in this study, we used Pichia pastoris expression system to construct an energy-saving, ecological and efficient way of preparing highly Bp-DNase I, which passes the way for the functional research.In present study, the sequence of Bp-DNase I gene was modified basing on preferred codons of P. pastoris and synthesized by overlapping PCR in vitro followed by secretorily expressing in P. pastoris GS115. The results showed that the yield of Bp-DNase I could reach about223.9mg/L, and the enzyme activity is23000U/mg, which is23times more than reported. The optimal pH and temperature of Bp-DNase I is7.9and37℃, respectively. The recombinant Bp-DNase I has high pH stability, and still has80%relatively activity after10min incubating in pH5-9buffers. In the process of producing in Pichia pastorios GS115, Bp-DNase I is modified by N-glycosylation, which was comfirmed by Endo H digestion. Meanwhile, it may be the reason of the deterioration of the thermal stability. All results of the above suggest that we can produce highly secretion expression of Bp-DNase I in the yeast expression system. Comparing with traditional way, this method is more efficient and more energy-saving.