Life Technologies Corporation

Invitrogen Corporation

Mary C Longo

Deb K Chatterjee

Elizabeth Flynn

Robert Oberfelder

Michael D Smith

Brian J Schmidt

Raymond D Harris

A John Hughes, Jr

Ilse I Blumentals

Roger S Lasken

The current invention provides methods for producing a polypeptide as inclusion bodies in bacterial host cells. The present methods are carried out by forming a gene construct comprising the genetic sequence encoding a polypeptide operatively linked to that of an inclusion partner protein, such that host cells comprising the gene construct produce the polypeptide as intracellular inclusion bodies thereby facilitating rapid isolation and purification of recombinant proteins. The invention further provides methods for producing protein molecular weight ladders for us in protein gel electrophoresis, as well as proteins and protein molecular weight ladders produced by these methods.

Methods for production of proteins

The current invention provides methods for producing a polypeptide as inclusion bodies in bacterial host cells. The present methods are carried out by forming a gene construct comprising the genetic sequence encoding a polypeptide operatively linked to that of an inclusion partner protein, such asthioredoxin or a modifiedthioredoxin, such that host cells comprising the gene construct produce the polypeptide as intracellular inclusion bodies. The methods of the present invention facilitate the rapid isolation and purification of recombinant proteins. In addition, the present methods may be useful for producing polypeptides or proteins which are small and are typically difficult to express, as well as those proteins that are toxic to host cells such as. The present invention also provides plasmids, vectors and host cells to be used in the present invention for production of polypeptides, and methods of production of polypeptides using these vectors and host cells. The invention further provides methods for producing protein molecular weight ladders for use in protein gel electrophoresis, as well as proteins and protein molecular weight ladders produced by these methods.

Methods of production of proteins

The current invention provides methods for producing a polypeptide as inclusion bodies in bacterial host cells. The present methods are carried out by forming a gene construct comprising the genetic sequence encoding a polypeptide operatively linked to that of an inclusion partner protein, such as thioredoxin or a modified thioredoxin, such that host cells comprising the gene construct produce the polypeptide as intracellular inclusion bodies. The methods of the present invention facilitate the rapid isolation and purification of recombinant proteins. In addition, the present methods may be useful for producing polypeptides or proteins which are small and are typically difficult to express, as well as those proteins that are toxic to host cells such as . The present invention also provides plasmids, vectors and host cells to be used in the present invention for production of polypeptides, and methods of production of polypeptides using these vectors and host cells. The invention further provides methods for producing protein molecular weight ladders for use in protein gel electrophoresis, as well as proteins and protein molecular weight ladders produced by these methods.

The present invention discloses the cloning and expression in Escherichia coli of the SstI/SacI restriction-modification system. The SstI methylase gene was cloned by methylase selection (the 'Hungarian Trick' and the orientation and boundaries of the gene were established. The level of methylation of the host was improved significantly by expressing the SstI methylase from the strong RPSL promoter. Using the SstI methylase clone as a probe, a chromosomal map was generated in the area of the gene for the SstI methylase. DNA downstream from the methylase was cloned and found to contain the gene for the SstI endonuclease. The orientation of the SstI endonuclease was established and a strong promoter was placed close to the SstI endonuclease gene by generating nested deletions. By these methods, an E. coli strain was constructed which produced high levels of SstI endonuclease. The SacI locus in the S. achromogenes chromosome was found to be identical to the SstI locus in S. stanford in all measured respects.

Cloned SstI/SacI restriction-modification system

The present invention discloses the cloning and expression in Escherichia coli of the NsiI restriction-modification system from Neisseria sicca, utilizing a two step protocol. Initial protection of the E. coli host with methylase expressed on a plasmid was required to stabilize a compatible plasmid carrying the endonuclease gene on a single DNA fragment. A chromosomal map was generated localizing the genes for NsiI methylase and endonuclease. An E. coli strain was constructed which produced high levels of NsiI endonuclease.

Cloned NsiI restriction-modification system

Cloned Ssti/SacI restriction-modification system

The invention relates to a substantially pure thermostable DNA polymerase. Preferably, the DNA polymerase has a molecular weight of about 95 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to cloning and expression of the DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes.

Thermophilic DNA polymerase

The present invention discloses the cloning and expression in Escherichia coli of the NsiI restriction-modification system from Neisseria sicca, utilizing a two step protocol. Initial protection of E. coli host with methylase expressed on a plasmid was required to stabilize a compatible plasmid carrying the endonuclease gene on a single DNA fragment. A chromosomal map was generated localizing the genes for NsiI methylase and endonuclease. An E. coli strain was constructed which produced high levels of NsiI endonuclease.

The present invention is directed to recombinant hosts which contain and express the AluI Type-II restriction endonuclease gene. The present invention is also directed to vectors or DNA molecules which contain the gene, and to methods of producing the AluI enzyme. One source of the enzyme is Arthrobacter luteus, although other microorganisms may be used to isolate restriction endonuclease isoschizomers of the invention.

Cloning and expression of AluI restriction endonuclease

The present invention is directed to recombinant hosts which contain and express the ClaI Type-II restriction endonuclease and/or modification methylase genes. The present invention is also directed to vectors or DNA molecules which contain these gene, and to methods of producing the enzymes. One source of these enzyme is Caryophanon latum, although other microorganisms may be used to isolate the restriction endonuclease isoschizomers and modification methylase isoschizomers of the invention.

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Germantown, MD, United States of America

Mary C Longo