A red blood cell (RBC) having an agent linked thereto, wherein the agent is linked to at least one endogenous, non-engineered membrane protein of the RBC by a sortase-mediated reaction, preferably by a sortase-mediated glycine conjugation and/or a sortase-mediated lysine side chain ε-amino group conjugation, which may occurring at least on glycine (n) and/or lysine ε-amino group at internal sites of the extracellular domain of at least one endogenous, non-engineered membrane protein, preferably n being 1 or 2, as well as the use of the RBC for delivering drugs and probes.

The present invention pertains to a novel architecture of non-ribosomal peptide synthases (NRPS). The invention provides artificial NRPS wherein the naturally occurring terminal condensation or thioesterase-domain is replaced by internal condensation or dual condensation/epimerization domains. Moreover, the present invention enables the portability of terminal condensation domains to unrelated NRPS in respect of peptide release of linear peptides. The replacement results in a product independent release of the synthesized product and therefore enables the rational design of NRPS. The invention provides the new NRPS, nucleic acids encoding them, methods for artificial NRPS generation, and methods for producing non-ribosomal peptides.

The present invention pertains to a novel architecture of non-ribosomal peptide synthases (NRPS). The invention provides artificial NRPS wherein the naturally occurring terminal condensation or thioesterase-domain is replaced by internal condensation or dual condensation/epimerization domains. Moreover, the present invention enables the portability of terminal condensation domains to unrelated NRPS in respect of peptide release of linear peptides. The replacement results in a product independent release of the synthesized product and therefore enables the rational design of NRPS. The invention provides the new NRPS, nucleic acids encoding them, methods for artificial NRPS generation, and methods for producing non-ribosomal peptides.

The present invention is broadly concerned with new in vitro glycosylation methods that provide rational approaches for producing glycosylated proteins, and the use of glycosylated proteins. In more detail, the present invention comprises methods of glycosylating a starting protein having an amino sidechain with a nucleophilic moiety, comprising the step of reacting the protein with a carbohydrate having an oxazoline moiety on the reducing end thereof, to covalently bond the amino sidechain of the starting protein with the oxazoline moiety, wherein the glycosylated protein substantially retains the structure and function of the starting protein. Target proteins include oxidase, oxidoreductase and dehydrogenase enzymes. The glycosylated proteins advantageously have molecular weights of at least about 7500 Daltons. In a further embodiment, the present invention concerns the use of glycosylated proteins, fabricated by the methods disclosed herein, in the assembly of amperometric biosensors.

Provided are an improved culture process for reducing unwanted culture byproducts when a target protein is produced in a glutamine-free cell culture medium, an improved culture process for producing a target protein in a glutamine-free cell culture medium, or an improved culture process for producing a target protein in a glutamine-free cell culture medium.

Provided are an improved culture process for reducing unwanted culture byproducts when a target protein is produced in a glutamine-free cell culture medium, an improved culture process for producing a target protein in a glutamine-free cell culture medium, or an improved culture process for producing a target protein in a glutamine-free cell culture medium.

A tandem DNA element capable of enhancing protein synthesis efficiency, in particular, the nucleic acid construct is formed by an IRES enhancer (such as ScBOI1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, KlBOI1) derived from eukaryotic cells (such as yeast), a Ω sequence, and a yeast-specific Kozak sequence in tandem. The use of the nucleic acid construct in a yeast-based in vitro biosynthesis system (such as a yeast-based in vitro protein synthesis system) can significantly improve protein synthesis efficiency.

A tandem DNA element capable of enhancing protein synthesis efficiency, in particular, the nucleic acid construct is formed by an IRES enhancer (such as ScBOI1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, KlBOI1) derived from eukaryotic cells (such as yeast), a Ω sequence, and a yeast-specific Kozak sequence in tandem. The use of the nucleic acid construct in a yeast-based in vitro biosynthesis system (such as a yeast-based in vitro protein synthesis system) can significantly improve protein synthesis efficiency.

The present invention is broadly concerned with new in vitro glycosylation methods that provide rational approaches for producing glycosylated proteins, and the use of glycosylated proteins. In more detail, the present invention comprises methods of glycosylating a starting protein having an amino sidechain with a nucleophilic moiety, comprising the step of reacting the protein with a carbohydrate having an oxazoline moiety on the reducing end thereof, to covalently bond the amino sidechain of the starting protein with the oxazoline moiety, wherein the glycosylated protein substantially retains the structure and function of the starting protein. Target proteins include oxidase, oxidoreductase and dehydrogenase enzymes. The glycosylated proteins advantageously have molecular weights of at least about 7500 Daltons. In a further embodiment, the present invention concerns the use of glycosylated proteins, fabricated by the methods disclosed herein, in the assembly of amperometric biosensors.

This invention relates generally to pharmaceutical compositions and preparations of circular polyribonucleotides and uses thereof.