Method for producing the Hinc II restriction endonuclease and methylase

Abstract

The present invention is directed to a method for cloning and producing the Hinc II restriction endonuclease by (1) introducing the restriction endonuclease gene from Haemophilus influenzae Rc into a host whereby the restriction gene is expressed; (2) fermenting the host which contains the vector encoding and expressing the Hinc II restriction endonuclease, and (3) purifying the Hinc II restriction endonuclease from the fermented host which contains the vector encoding and expressing the Hinc II restriction endonuclease activity.

What is claimed is: 
     
       1. Isolated DNA coding for the HincII restriction endonuclease, wherein the isolated DNA is obtainable from the vector p(pUC)HincIIRM-10-5.7. 
     
     
       2. A recombinant DNA vector comprising a vector into which a DNA segment coding for the HincII endonuclease produced by Haemophilus influenza Rc ATCC No. 53876 has been inserted. 
     
     
       3. Isolated DNA coding for the HincII restriction endonuclease and methylase, wherein the isolated DNA is obtainable from the vector p(pUC)HincIIRM-10-5.7. 
     
     
       4. A cloning vector which comprises the isolated DNA of claim 3. 
     
     
       5. The cloning vector of claim 4, wherein the cloning vector comprises p(pUC)HincIIRM-10-5.7. 
     
     
       6. A host cell transformed by the vector of claim 2, 4 or 5. 
     
     
       7. A method of producing HincII restriction endonuclease comprising culturing a host cell transformed with the vector of claim 2, 4 or 5 under conditions suitable for the expression of said endonuclease. 
     
     
       8. A method of cloning a HincII restriction endonuclease gene which comprises: (a) forming a library from plasmid DNA from Haemophilus influenza Rc obtainable from ATCC No. 53876 by digesting said plasmid DNA with Bgl II;   (b) contacting the plasmid library of step (a) with Hinc II to form a digestion pool, transforming the digestion pool into a host cell, and screening for cloning vectors which contain DNA coding for the HincII methylase;   (c) purifying HincII restriction-endonuclease from the Haemophilius influenza Rc of step (a), partially sequencing the endonulcease and forming a DNA probe based on the partial amino acid sequence of the endonuclease;   (d) determining the direction and location of DNA coding for the endonuclease by contacting the probe of step (c) with the cloning vectors of step (b) containing DNA coding for the HincII methylase;   (e) from the direction and location of the DNA coding for the endonuclease determined in step (d), forming a library which contains DNA coding for the HincII endonulease; and   (f) isolating one or more cloning vectors of step (e) which contain DNA coding for the HincII endonuclease.   
     
     
       9. A method for producing HincII restriction endonuclease comprising culturing a host cell transformed with the cloning vector of claim 8, step (f) under conditions suitable for expression of said endonuclease. 
     
     
       10. The isolated DNA of claim 1 or 3, wherein the isolated DNA includes the DNA sequence: .[.TAC TCA AAG TAT TTT GGA TAA ATA GTC CTA TAA TTG NNA.]. .Iadd.ATG AGT TTC ATA AAA CCT ATT TAT CAG GAT ATT AAT TCA .Iaddend.ATA TTA ATC GCG CAA AAA GTG AAA CGT CCT AAA TCA GGT ACT CTG TCA GGT CAT GCT GCA GGG GAA CCA TTT GAA AAA TTA GTA AAG TTT TTG AAA GAA AAC CTG TCA GAT TTA ACA TTT AAG CAA TAT GAA TAT CTT AAT GAT TTA TTT ATG AAG AAC CCT GCG ATA ATT .[.GAG.]. .Iadd.GGA .Iaddend.CAT G 3'. .Iadd. 
     
     
       11.  A method for producing a Hinc II recombinant restriction endonuclease which recognizes the following base sequence in double-stranded DNA molecules:   GTPyPuAC     and cleaves between the Py and Pu of said sequence, comprising culturing a host cell transformed with a cloning vector under conditions suitable for expression of said endonuclease, wherein the cloning vector comprises isolated DNA obtainable from Haemophilus influenzae Rc. .Iaddend. .Iadd.   
     
     
       12.  The method of claim 11, wherein said cloning vector comprises isolated DNA which includes the DNA sequence: ATG AGT TTC ATA AAA CCT ATT TAT CAG GAT ATT AAT TCA ATA TTA ATC GCG CAA AAA GTG AAA CGT CCT AAA TCA GGT ACT CTG TCA GGT CAT GCT GCA GGG GAA CCA TTT GAA AAA TTA GTA AAG TTT TTG AAA GAA AAC CTG TCA GAT TTA ACA TTT AAG CAA TAT GAA TAT CTT AAT GAT TTA TTT ATG AAG AAC CCT GCG ATA ATT GGA CAT G 3'. .Iaddend. .Iadd. 
     
     
       13.  A method for producing a Hind II recombinant restriction endonuclease which recognizes the following base sequence in double-stranded DNA molecules:   GTPyPuAC     and cleaves beween the Py and Pu of said sequence, comprising culturing a host cell transformed with a cloning vector under conditions suitable for expression of said endonuclease, wherein the cloning vector comprises isolated DNA obtainable from Haemophilus influenzae Rd. .Iaddend.