Study Of The Method To Highly-Express Exogenous Protein In E.coli And It's Application To Natto Kinase And Pro-Natto Kinase

Aims: To improve exogenous protein expression in E.coli, a new method was studied and applied to Nattokinase and pro-Nattokinase. We usually clone exogenous proteins using regular methods of molecular cloning, but the expression rate is often too low and even no expression. In the large-scale production of exogenous proteins, some high-expression strains must be established. In this experiment, we studied most of the factors influencing the protein expression, and then designed a new method of molecular-cloning.Methods: Among all of the factors influencing expression, we focused on two factors: the Free Energy of the Translation Initiation Region (TIR) and the Codon Preference of E.coli. During the cloning, there are two principles of increasing the A G?of the TIR region and adopting more preferred codons. The A G?of the TIR region is mainly decided by the content of G+C in TIR. In general, the less content of G+C, the higher A G?of the TIR region. Based on the two principles mentioned above and the trait of wobbling bases of triplet code, we mutated the wobbling bases of the head 35 bases of pro-NK and NK cDNA. When contradiction occurred based on the two principles in some sites, we randomly changed them, and it would result in varied combinations in the randomly changed sites. Computer software DNASIS 2.5 was utilized to analyse the A G?of all the sequences. The first ten sequence with the high A G were chosen for subsequent cloning. Using synthetic primers containing the changed sites, ten sequences with different wobbling sites were amplified by PCR and then cloned to pET3c respectively. E.coli strain BL21(DE3) was transformed and induced to express recombinant NK. It was proved by SDS-PAGE that the highest expression rate was above 30%. The target proteins existed as inclusion body and were recovered. Then the proteins were purified by Superdex-200 gel-filtrationchromatography. The enzyme activity of nattokinase and pro-nattokinase was determinated by CLT method. The recombinant with the highest expression rate was sequenced. Results:1. We obtained high-expression strains for both pro-NK and NK. From the ten clone plates, we picked clones and analysed them. For pro-NK, there were 1 clone whose expression rate was above 30%; for mature NK, there were 3 high-expression clones whose expression rate was above 30%.2. The proteins were purified by gel-filtration chromatography. The purity reached about 96%.3. The enzyme activity of the pro-Nattokinase solution after ultra-filtration was about 150U/ml, and that of the Nattokinase solution was 220U/ml.Conclusion: Codon preference and the A G?of the TIR region were important factors influencing the expression rate. The reduction of G+C content will decrease the stability of secondary structure. According to these principles, the TIR was mutated and the resulted sequences were analysed using DNASIS 2.5. Ten primers were designed and cloned. This should be a rapid and reliable method to obtain high expression strain of exogenous protein.