University of Wyoming

Other

The Texas A&m University System

Johns Hopkins University

Human Genome Sciences, Inc.

University of Notre Dame Du Lac

Glycobac, LLC

Donald L Jarvis

Christoph Geisler

Linda A Guarino

John Scott T Jaques

Timothy A Coleman

Shawn M Lawrence

Michael J Betenbaugh

Yuan C Lee

Malcolm J Fraser

James C Carrington

Hideaki Mabashi-Asazuma

Ajay Maghodia

The current teachings are directed to virus free cells lines derived from virus-contaminated starting material, such as an organism or a cell line. Methods for obtaining virus free cell lines obtained from virus-contaminated starting material are also provided. Exemplary virus free cell lines include: cell lines derived from acell line contaminated with Sf-rhabdovirus, wherein the cell lines lack Sf-rhabdovirus; and cell lines derived from acell line contaminated with an alphanodavirus, wherein the cell line lacks an alphanodavirus.

Virus-free cell lines and methods for obtaining same

Compositions for reducing fucosylation of glycoproteins in insect cells are provided. Also disclosed are methods of use of such compositions for the production of recombinant humanized proteins.

Compositions and methods for reducing fucosylation of glycoproteins in insect cells and methods of use thereof for production of recombinant glycoproteins

The present invention relates to methods of facilitating the expression of recombinant polypeptides from cells, extracellular fluids, extracellular fibers, or any combination thereof, obtained from transgenic insect cells and larvae comprising a bacterial GlcNAc-6-P 2'-epimerase (GNPE), which is capable of converting N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) to N-acetyl-D-mannosamine-6-phosphate (ManNAc-6-P). The invention relates to methods to promote efficient glycoconjugate sialylation, by providing simpler ways to produce large intracellular pools of sialic acid precursors. The invention is also directed to nucleic acids, vectors, and cells comprising nucleic acids encoding polypeptides involved in the synthesis of sialic acid precursors, and cells in combination with nucleic acids encoding glycosyltransferases, including sialyltransferases, to facilitate the production of humanized recombinant glycoproteins. The engineered cells can be used to produce glycosylated proteins lepidopteran insects and cultured cell lines derived from, and silkworms, such as, particularly those that can be infected by baculovirus expression vectors.

Transgenic insect cells comprising a bacterial GlcNAc-6-P 2'-epimerase

A transgenic silkworm system for recombinant glycoprotein production is provided.

Production of human glycosylated proteins in silk worm

A transgenic insect cell line for production of recombinant glycoproteins possessing sulfated, complex N-glycans is provided.

Insect cell line for production of recombinant glycoproteins with sulfated complex N-glycans

A transgenic insect cell line for production of elevated levels of recombinant glycoproteins comprising mammalian-like N-glycans is provided. Also disclosed are nucleic acid sequences encoding &#x3b2;-N-acetylglucosaminidases.

Lepidopteran insect N-acetylglucosaminidase genes and their use in glycoengineering

The present invention relates to new methods to promote sialylation of glycoconjugates, including recombinant glycoproteins, in glycoconjugate production systems. The invention relates to methods to promote efficient glycoconjugate sialylation in recombinant expression systems, by providing simpler and more economical ways to produce large intracellular pools of sialic acid precursors. The invention is directed to nucleic acids, vectors, and cells harboring vectors comprising nucleic acids encoding enzymes involved in the synthesis of sialic acid precursors, and cells harboring these nucleic acids in combination with nucleic acids encoding glycosyltransferases, including sialyltransferases, to facilitate the production of humanized recombinant glycoproteins in bacterial, fungal, plant, and animal cell expression systems. The engineered cells can be used to produce glycosylated proteins in virally-infected, transiently-transformed, or stably-transformed host cells, including lepidopteran insects and cultured cell lines derived from, andthat can be infected by baculovirus expression vectors.

Methods of using a bacterial GlcNAc-6-P 2'- epimerase to promote sialylation of glycoconjugates

Methods for manipulating carbohydrate processing pathways in cells of interest are provided. Methods are directed at manipulating multiple pathways involved with the sialylation reaction by using recombinant DNA technology and substrate feeding approaches to enable the production of sialylated glycoproteins in cells of interest. These carbohydrate engineering efforts encompass the implementation of new carbohydrate bioassays, the examination of a selection of insect cell lines and the use of bioinformatics to identify gene sequences for critical processing enzymes. The compositions comprise cells of interest producing sialylated glycoproteins. The methods and compositions are useful for heterologous expression of glycoproteins.

Engineering intracellular sialylation pathways

The invention includes a nucleic acid having a sequence at least 98% homologous to SEQ ID NO: 1, which encodes the &#x3b1; subunit of canine thyroid stimulating hormone (TSH). The invention also includes a nucleic acid having a sequence at least 98% homologous to SEQ ID NO: 2, which encodes the &#x3b2; subunit of canine TSH. The invention also includes a method of producing a recombinant canine thyroid stimulating hormone (rcTSH) subunit by expressing a nucleic acid having a sequence of SEQ ID NO: 1 and a nucleic acid having a sequence of SEQ ID NO: 2 in a transgenic insect cell modified to sialylate proteins and producing a sialylated rcTSH subunit. The insect cell may be a lepidopteran cell. The rcTSH may be used for diagnosis and treatment. It may be used to diagnose canine hypothyroidism.

Recombinant canine thyroid stimulating hormone and methods of production and use thereof

The invention includes a nucleic acid having a sequence at least 98% homologous to SEQ ID NO: 1, which encodes the &#x3b1; subunit of canine thyroid stimulating hormone (TSH). The invention also includes a nucleic acid having a sequence at least 98% homologous to SEQ ID NO: 2, which encodes the &#x3b2; subunit of canine TSH. The invention also includes a method of producing an recombinant canine thyroid stimulating hormone (rcTSH) subunit by expressing a nucleic acid having a sequence of SEQ ID NO: 1 and a nucleic acid having a sequence of SEQ ID NO: 2 in a transgenic insect cell modified to sialylate proteins and producing a sialylated rcTSH subunit. The insect cell may be a lepidopteran cell. The rcTSH may be used for diagnosis and treatment. It may be used to diagnose canine hypothyroidism.

Disclosed are a variety of recombinant baculovirus vectors, and host insect cells, which comprise at least one oligosaccharide processing enzyme gene. The vectors and cells may optionally comprise other heterologous structural genes, including further protein processing enzymes. Methods of making and using the recombinant baculoviruses and vectors are provided, including their uses in recombiant protein production and as insecticides.

Modifying insect cell gylcosylation pathways with baculovirus expression vectors

The present invention provides an method for isolating and purifying recombinantly produced proteins. This invention involves the use of an expression vector which includes a nuclear targeting signal sequence which effectively directs newly synthesized proteins to the nucleus; a cleavage recognition sequence which cleaves specifically at a pre-determined cleavage site after addition of a viral enzyme; and a cDNA sequence which codes for a desired protein. Specifically the production and isolation of a desired protein is accomplished through the use of lepidopteran cells transfected or infected with recombinant baculovirus expression vector comprising a polyhedrin gene derived nuclear targeting signal sequence and a cleavage recognition sequence derived from a potyvirus polyprotein. The newly synthesized proteins are directed into the nucleus whereupon nuclear protein is extracted from the nucleus. Thereafter the desired protein is bound to an affinity matrix embedded with antibodies directed against the nuclear targeting sequence. Afterwards the desired protein is cleaved from the affinity matrix with the addition of a viral enzyme.

Method and vector for the purification of foreign proteins

This invention details a novel and unique plasmid vector for the dual purposes of either producing transformed insect cell clones or recombinant baculoviruses. The transformed insect cell clones will continuously and permanently produce an efficiently processed desired foreign gene product. The recombinant baculoviruses will transiently express the desired foreign gene during immediate early phase of infection. This unique vector employs promoters from two different immediate early baculovirus genes along with the natural occurring polyhedrin promoter and gene. This unique combination creates a situation where the virus is highly infectious in vivo and resistant to inactivation in nature, because it is occlusion positive, and expresses the foreign gene product during immediate early phase of infection. Therefore this virus would be very effective as a delivery system of pesticides.

Use of baculovirus early promoters for expression of foreign genes in

The present invention provides an alternative strategy for baculovirus-mediated foreign gene expression: the promoters from immediate-early or delayed-early baculovirus viral genes were used to obtain the continuous expression of foreign genes in stably-transformed insect cells. The IE1 or the 30K promoters from Autographa californica nuclear polyhedrosis virus can drive the continuous expression of a foreign gene in insect cells. IE1-.beta.-galactosidase or 39K-.beta.-galactosidase constructs were used in combination with the IE1-neomycin resistan

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Laramie, WY, United States of America

Donald L Jarvis