New England Biolabs, Inc.

James C Samuelson

Shuang-Yong Xu

Minyong Chen

Christopher H Taron

Ming-Qun Xu

Xiaofeng Shi

Margaret Heider

Aihua Zhang

Pingfang Liu

Geoffrey G Wilson

Theodore B Davis

Elisabeth A Raleigh

John J Pelletier

Maurice W Southworth

Marion Sibley

Carine Robichon-Iyer

Jianying Luo

Diana O'Loane

Ming-Oun Xu

Provided herein is a thermolabile proteinase and methods of using the same. In some embodiments, the thermolabile proteinase may comprise an amino acid sequence that is at least 90% identical to any of SEQ ID NOs:1-11 and at least one amino acid substitution in helix 3. The thermolabile proteinase is active at a temperature in the range of 4&#xb0; C.-40&#xb0; C. and is inactivated by raising the temperature to above 50&#xb0; C., where the proteinase is substantially inactive at 65&#xb0; C.

Proteinases with improved properties

Methods of capturing N-glycan linked glycomolecules including N-glycans, N-glycopeptides and N-glycoproteins are described. The methods provide substantially unbiased capture of charged and uncharged N-glycans and/or N-glycan linked glycomoleules. Binding reagents for substantially unbiased binding of N-glycans and/or N-glycan linked glycomolecules are also described.

Universal N-glycan binding reagent

Methods of capturing N-glycan linked glycomolecules including N-glycans, N-glycopeptides and N-glycoproteins are described. The methods provide substantially unbiased capture of charged and uncharged N-glycans and/or N-glycan linked glycomolecules. Binding reagents for substantially unbiased binding of N-glycans and/or N-glycan linked glycomolecules are also described.

Compositions relating to a combination of two types of separation matrix; and to variant host cells which contain at least one essential host protein that is fused to an affinity binding tag or has been mutated to replace at least two of a plurality of histidines or basic amino acids are provided. Methods are also provided that relate to isolating a recombinant protein from a lysate.

Composition, method and kit for obtaining purified recombinant proteins

Compositions and methods are provided for expression of a toxic protein in a host cell preferably a bacterial host cell where at least one T7 RNA polymerase gene Is contained on the host cell chromosome and one or more genes encoding a T7 RNA polymerase inhibitor is located on an F' plasmid or on the chromosome.

Expression of toxic genes in vivo in a non-natural host

Methods are provided for engineering novel strand-specific nicking endonucleases by means of an in vivo enrichment of a plasmid library containing a randomly mutagenized restriction endonuclease gene. The plasmids contain adjacent to the gene a cleavable or nickable sequence for cleaving or nicking by the endonuclease product of the gene and a second recognition site for a second endonuclease. The plasmid library is used to transform unmodified host cells. Plasmids from the cultured transformed cells may be analyzed by an in vitro assay for nicking and the nicked plasmids pooled and used to transform host cells. The product is then pooled and the single-stranded specificity of the endonuclease is then determined. The product is either cloned after amplification or identified by use of a selectable marker.

Method for engineering strand-specific nicking endonucleases from restriction endonucleases

Methods and compositions are provided for altering the DNA recognition and cleavage characteristics of an endonuclease without prior knowledge of the endonuclease's three-dimensional structure and/or amino acid residues responsible for activity and/or specificity. Methods include subjecting a mutagenized endonuclease gene library to a genetic selection in prokaryotic cells which tolerate the expression of mutated endonuclease and where the endonuclease is active and determining the altered recognition-site specificity for the endonuclease.

Alteration of restriction endonuclease specificity by genetic selection

The present invention relates to recombinant DNA encoding the AcuI restriction endonuclease as well as AcuI methylase, and expression of AcuI restriction endonuclease and AcuI methylase incells containing the recombinant DNA.

Method for cloning and expression of AcuI restriction endonuclease and AcuI methylase in E. coli

The present invention relates to recombinant DNA encoding the PpuMI restriction endonuclease as well as PpuMI methylase, expression of PpuMI restriction endonuclease and PpuMI methylase in cells containing the recombinant DNA.

Method for cloning and expression of PpuMI restriction endonuclease and PpuMI methylase in E. coli

The present invention relates to recombinant DNA which encodes the BstYI restriction endonuclease as well as BstYI methyltransferase, expression of BstYI restriction endonuclease and M.BstYI in cells containing the recombinant DNA. It also relates to methods for purification of the recombinant BstYI restriction endonuclease and BstYI methyltransferase.

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Newburyport, MA, United States of America

James C Samuelson