Cloning And Expression Of ΦHAU3～R Gene Of Streptomyces Lividans 1326

HAU3R gene of S. lividans 1326 is directly related to its resistance to actinophage HAU3. The deduced amino acid sequence of HAU3R protein suggested that propablely it is a kind of endonuclease similar to Ea59. This study mainly focused on over-expression of HAU3R gene in E. coli and Streptomyces.The 1.4kb HAU3R gene, which is from Strepyomyces lividans 1326, was amplified by PCR. And the PCR product was cloned downstream of promoter PT7 of pET-28a (+), a Escherichia coli expression vector, then the counterpart of HAU3R gene cloned in pET-28a (+) was replaced by a 1.3kb NcoI-EcoRI fragment from the template used for PCR above. A resulted recombinant plasmid, pHZ2055, was obtained. The HAU3R gene was over-expressed when E. coli BL21 (DE3)(pHZ2055) was induced with IPTG, but the products existed in the form of inclusion bodies. In order to know whether the HAU3R gene amplified by PCR had its natural activities, HAU3R gene from pHZ2055 was cloned into pHZ1060, a Streptomyces-E. coli bifunctional expression vector, to give pHZ2057. When E. coli DH5 a (pHZ2057) was induced at 42C, large amounts of HAU3R protein were produced to form inclusion bodies. When pHZ2057 was introduced into S. lividans ZX1, it could confer partial resistance to actinophage HAU3 on S. lividans ZX1. To express HAU3R gene as fusion protein, it was cloned into pGEX-KG and fused with GST, which is controlled by promoter Ptac. Unfortunately, the fusion protein was insoluble, either. At last, HAU3R gene was inserted downstream of PtipA of pIJ6021, a Streptomyces expression vector. The final construct was designated as pHZ2085. The expression of HAU3R gene was leaky in S. lividans ZX57, and HAU3R gene was highly expressed when ZX57 (pHZ2085) was induced with thiostrepton at the concentration of 5 g/ml. The product was soluble.Additionally, HAU3R gene with it own promoter was cloned into high-copy plasmid pIJ653 and integrative plasmid pSET152, respectively. Transformants of S. lividans ZX1 carrying these clones were infected with HAU3, respectively, but the results shown that there was no significantcorrelation between the copy number of HAU3R gene and the level of resistance to HAU3.