RESUMO
Expression of recombinant full length human alpha 2,6(N)sialyltransferase has been scaled-up in S. cerevisiae in a 150-l bioreactor yielding 47 U at a concentration of 0.31 U/l. The protein specific activity as measured in reconstituted yeast lyophilisate was 0.8 mU/mg protein. The recombinant enzyme exhibited similar Michaelis constants as previously determined for the native rat enzyme. By immunoblotting the enzyme was shown to be heterogeneous by size (44-48 kD) and N-glycosylated. We conclude that recombinant alpha 2,6(N)sialyltransferase expressed in S. cerevisiae is retained in the endoplasmic reticulum as a fully active enzyme.
Assuntos
Sialiltransferases/biossíntese , Western Blotting , Humanos , Cinética , Peso Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Sialiltransferases/metabolismoRESUMO
We report in this communication the first large-scale heterologous expression of a glycosyltransferase in yeast. A soluble form of a human beta-1,4-galactosyltransferase (EC 2.4.1.38) was expressed using a Saccharomyces cerevisiae expression system. Fermentation technology afforded the means to increase the expression level of the beta-1,4-galactosyltransferase up to a concentration of 700 mU/liter. The enzyme was produced at a scale of 200 units. The recombinant soluble enzyme was purified 766-fold to a specific activity of approx. 2 U/mg using a purification protocol based on sequential affinity chromatography on N-acetylglucosaminyl- and alpha-lactalbumin-Sepharose, respectively. This study demonstrates that heterologous expression of a glycosyltransferase is possible on a large scale and offers an alternative to natural sources like human breast milk or bovine colostrum.
Assuntos
N-Acetil-Lactosamina Sintase/genética , Saccharomyces cerevisiae/genética , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/genética , Humanos , N-Acetil-Lactosamina Sintase/biossíntese , N-Acetil-Lactosamina Sintase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Sequences coding for the cytoplasmic and transmembrane domains were removed from the cDNA of the human Golgi resident membrane protein beta 1,4 galactosyltransferase (gal-T). The remaining sequences coding for the stem and catalytical domains of this glycosyltransferase were fused to sequences coding for the yeast invertase signal sequence. The hybrid was inserted together with a constitutive yeast promoter and a terminator into a E. coli/yeast shuttle vector. Saccharomyces cerevisiae strain BT150 transformed with this new expression vector expressed enzymically active soluble enzyme, whereas no activity was detectable in mock-transformed yeasts. The enzyme product was identified by HPLC analysis and shown to correspond to the expected product N-acetyllactosamine.
Assuntos
Galactosiltransferases/genética , Sequência de Bases , Clonagem Molecular , Galactosiltransferases/química , Expressão Gênica , Humanos , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genética , Proteínas Recombinantes , Saccharomyces cerevisiae/enzimologia , Solubilidade , Relação Estrutura-AtividadeRESUMO
Our department is committed to a process of continuous quality improvement focusing on delivering the best possible pharmaceutical care services. Three committees, each with representation from pharmacists and pharmacy technicians from all areas of the department, were convened in 1991 to 1992 to further identify areas for service enhancement and to plan for the future. Based on the recommendations of these committees, further expansion in ambulatory services is a priority. Other changes will include further automation of the drug-distribution system, examination of the role of the pharmacy technician, development of an automated patient-care system with direct physician order entry, computerized documentation of clinical interventions, and reaffirmation of the role of the pharmacist as the professional responsible for proper use of medications at UMHC.
