Roche Diagnostics Operations, Inc.

Roche Molecular Systems, Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Michael Greif

Harald Sobek

Marco Thomann

Sebastian Malik

Rainer Mueller

Johann-Peter Thalhofer

Waltraud Ankenbauer

Alfred Engel

Frank Laue

Bernhard Suppmann

Manfred Schmidt

Christine Jung

Christian Rudolph

Helmut Schwab

Bernd Nidetzky

Ulrike Fischer

Armin Ruf

Christiane Luley

Doris Ribitsch

Sabine Zitzenbacher

Katharina Schmoelzer

Tibor Czabany

The properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions are disclosed. Particularly, mutants that exhibit &#x3b1;-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site, are disclosed. A fundamental finding documented in the present disclosure is that enzymes are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

CMP-dependent sialidase activity

The present disclosure is directed to the properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions. Any of the mutants disclosed in here exhibit alpha-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site. A fundamental finding documented in the present disclosure is that these enzymes are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

Aqueous composition

The present invention relates to a formulation of a thermostable DNA polymerase which is completely free of detergents and its particular use in real time polymerase chain reaction (PCR). Such a formulation may be obtained if the selected purification method does not require the addition of a detergent at any purification step.

Detergent free polymerases

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. Contrary to previous findings certain truncations were found to exhibit sialidase enzymatic activity, particularly a variant of human sialyltransferase (hST6Gal-I) with a truncation deletion involving the first 89 N-terminal amino acids of the respective wild-type polypeptide. A fundamental finding documented in the present disclosure is that there exists a variant of this enzyme which is capable of catalyzing transfer of a glycosyl moiety as well as hydrolysis thereof. Thus, disclosed is a specific exemplary variant of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variant of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Quantitative control of sialylation

The present disclosure provide novel variants of T7 RNA polymerase. Embodiments of T7 variants, according to the instant invention, include a Cysteine-Serine substitution on position 723 of the amino acid sequence of the T7 polypeptide. Embodiments of T7 variants according to the instant invention have a DNA-dependent RNA polymerase enzymatic activity and a reduced tendency to form intramolecular homodimers by way of oxidizing thiol groups. The amino acid substitutions within the T7 variants disclosed herein impact minimally, if at all, the RNA polymerase activity of the T7 polypeptide. Further, the mutations of the disclosed embodiments may optionally be combined with mutations which provide enhanced thermostability compared to the wild-type reference.

T7 RNA polymerase variants with cysteine-serine substitutions

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. It was found that the combination of two different truncation variants of human &#x3b2;-galactoside-&#x3b1;-2,6-sialyltransferase I (hST6Gal-I) exhibited different specific sialyltransferase enzymatic activities. In one example, under conditions wherein the first variant &#x394;89 hST6Gal-I catalyzed formation of bi-sialylated target molecules the second variant &#x394;108 hST6Gal-I catalyzed formation of mono-sialylated target molecules. Thus, disclosed are variants of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variants of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

Quantitative control of sialylation and specific mono-sialylation

The present invention provides improved variants of T7 RNA polymerase by introducing novel mutations which lead to improved thermostability of the enzyme. According to the invention, amino acid substitutions at the positions Val426, Ser633, Val650, Thr654, Ala702, Val795, and combinations thereof are advantageous.

T7 RNA polymerase variants with enhanced thermostability

A purified thermostable enzyme is derived form the thermophilic archaebacterium. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3'-5&#x2032; exonuclease and cleaves to produce 5&#x2032;-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Taq especially for nucleic acid amplification by the polymerase chain reaction (PCR).

Thermostable enzyme promoting the fidelity of thermostable DNA polymerases&#x2014;for improvement of nucleic acid synthesis and amplification in vitro

The invention relates to a composition comprising a first modified thermostable enzyme exhibiting 3'exonuclease activity but essentially no DNA polymerase activity and a second modified thermostable enzyme exhibiting DNA polymerase activity, whereas the fidelity of an amplification process is enhanced by the use of the composition in an amplification process in comparison to the use of the single second enzyme in an amplification process and, whereas said first and said second modified thermostable enzyme is reversibly modified by an inhibiting agent which results in essentially complete inactivation of enzyme activity, wherein incubation of said first and said second modified thermostable enzyme in an aqueous buffer at alkaline pH at a temperature less than 25&#xb0; C. for 20 minutes results in no significant increase in the activity of said first and said second modified thermostable enzyme, wherein incubation at a temperature greater than 50&#xb0; C. in an aqueous buffer at alkaline pH results in at least tow-fold increase in enzyme activity in less than 20 minutes which allow formation of primer extension products.

Reversibly modified thermostable enzymes for DNA synthesis and amplification in vitro

A purified thermostable enzyme is derived from the thermophilic archaebacterium. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3'&#x2013;5&#x2032; exonuclease and cleaves to produce 5&#x2032;-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Tag especially for nucleic acid amplification by the polymerase chain reaction (PCR).

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Penzberg, Germany

Michael Greif