Methods and compositions relating to the engineering of an improved protein with methionine--lyase enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine--lyase (CGL) comprising one or more amino acid substitutions and capable of degrading methionine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with methionine depletion using the disclosed proteins or nucleic acids.

The present invention relates to the production of adeno-associated viral vectors in insect cells. The insect cells therefore comprise a first nucleotide sequence encoding the adeno-associated virus (AAV) capsid proteins, whereby the initiation codon for translation of the AAV VP1 capsid protein is an AUG. Upstream of the VP1 open reading frame an alternative out of frame start codon is placed such that translation initiation of the VP1 protein is modified, i.e. reduced, to allow production of VP1:VP2:VP3 in a good stoichiometry resulting in AAV with high potency.

The present invention provides methods, compositions, and kits for preparing and using adeno associated virus and baculovirus. The methods for producing adeno associated virus and baculovirus particles include using histidine rich peptides and other cationic peptides as transfection reagents. The adeno associated virus are pseudotyped with capsids, in particular for use in gene therapy and/or diagnostics. The baculovirus are also used to prepare adeno associated virus.

Methods of purifying a virus from a virus-infected cell lysate using three-phase partitioning (TPP) are disclosed. The methods comprise a first round of TPP, including mixing a cell lysate comprising a virus with ammonium sulfate and t-butanol, and separating the mixture, thereby forming a first aqueous phase, a first organic phase, and a first interphase. The first aqueous phase can comprise the virus, which can be subjected to a second round of TPP, resulting in a second aqueous phase, a second organic phase, and a second interphase. The second interphase can comprise highly purified virus. The methods can comprise subjecting a first aqueous phase to further purification by column chromatography or density gradient centrifugation. Purification of AAV, including AAV2, AAV5 and AAV6, from lysates of infected insect cell cultures is demonstrated. TPP-purified AAV particles infect at least as well as those prepared by standard methods.

The invention pertains to the technical fields of genetic engineering and cell engineering, and in particular relates to a rhabdovirus-negative Spodoptera frugiperda insect cell line, and screening, identification and application thereof. According to the invention, the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9, with a CCTCC accession number C202246, is obtained through screening and identification. The cell line is verified through various high-sensitivity test methods such as nested PCR, transcriptome next-generation sequencing, real-time fluorescence quantitative PCR and TAQMAN probe-based real-time PCR, and finally obtained the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9. The cell is tested for sterility, mycoplasma, exogenous virus and tumorigenicity according to pharmacopeial requirements, and the results show that all indicators satisfy the requirements, and the cell can be used in or for the production of recombinant proteins and recombinant protein vaccines based on the baculovirus expression system.

The present invention relates to a method for producing a single cell clone of an insect cell comprising integrated into the genome of the cell at least one expression cassette for inducible expression of parvoviral Rep 78 and 52 proteins. The insect cells can be used for the production of parvoviral vectors by transfection constructs comprising parvoviral Cap and ITR sequences. Single cell clones obtained in the methods of the invention can be expanded for into a cell bank for the production of clinical grade parvoviral gene therapy vectors.

A method of easily producing an immunogenic pupa for oral administration and a pupa for oral administration produced by the method are provided. The method is a method of producing a pupa for oral administration, including a step of infecting a larva or pupa of an insect capable of being baculovirus-infected with a recombinant baculovirus having DNA encoding an antigen protein introduced therein and freeze-drying a pupa having pupated from the infected larva or the infected pupa.

An insect culture maintenance system includes a sequence of open-ended cylindrical tubes [500, 502, 504, 506] joined pairwise alternately at their tops and bottoms using multiple dual-cap connectors. Each dual-capped connector has a channel from an inside of a first cap to an inside of a second cap. In use, connectors that cap the bottoms of the tubes [508, 512] are filled with insect food media [518, 520], while connectors that cap the tops of the tubes [510] are open. As a result of this design, adults pass from one tube to the next through the top dual-cap connectors, while larvae pass from one tube to the next through the bottom dual-cap connectors, resulting in propagation of subsequent generations of insects through the sequence of tubes.

The present disclosure relates to a method of obtaining a cell where fucosylation pathways are modified, leading to production of partially fucosylated and non-fucosylated protein products, specifically antibodies from the cell. The present disclosure employs the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. The method of the present disclosure targets the Fut8 gene and GMD gene in a cell. Such products are used in developing therapeutics and biomarkers, and in diagnosis and prognosis of diseases.

Methods and compositions relating to the engineering of an improved protein with methionine--lyase enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine--lyase (CGL) comprising one or more amino acid substitutions and capable of degrading methionine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with methionine depletion using the disclosed proteins or nucleic acids.