E. coli Poly(A) Polymerase

Cat. No.: E406458

GRADE & PURITY PharmPure™, Pharmaceutical grade

Descripción general

The introduction of Poly A tail at the 3'end of mRNA by tailing enzyme can increase the stability of mRNA and increase the efficiency of mRNA translation. The advantage of using the tailing enzyme to introduce the Poly A tail is that it is simple and easy to implement, avoiding the instability of the plasmid caused by the introduction of too many consecutive T bases during plasmid construction.

E. coli Poly (A) Polymerase does not rely on the presence of a template, and can catalyze the incorporation of ATP into the 3´ end of RNA in the form of AMP, that is, a poly A tail is added to the 3´ end of RNA. Poly (A) polymerase has a high tailing efficiency and can add 20~200 A bases to the 3´ end of RNA. Poly (A) structure helps to improve the translation efficiency of mRNA.

This product is expressed in large-scale fermentation using Escherichia coli, is produced with medicinal specifications of raw materials, and strictly controls host protein residues, nucleic acid residues, etc., and conforms to GMP standard product production and quality management procedures to ensure that the production process and all raw materials can be traced.

Quality requirements

Project	Standard	Method
Exterior	Clear liquid	Visual inspection
Visible foreign body	Compliance	Chinese Pharmacopoeia 2020 Edition Fourth Part 1 Lamp Inspection Method (General Rule 0904)
pH value	7.0-8.0	Chinese Pharmacopoeia 2020 Edition Part IV pH Determination Method (General Principle 0631)
Active	4.9KUml-5.1KU/ml	Tailing modification and efficiency determination method
Purity	≥95%	Chinese Pharmacopoeia 2020 Edition Part IV High Performance Liquid Chromatography (General Principle 0512)
Endonuclease residue	004-DNA degradation does not exceed 10%	Incubate SU enzyme with 004-DNA.37C for 3h
Exonuclease residue	019 HindⅢ-DNA degradation does not exceed 10%	Incubate SU enzyme with 019 HindⅢ-DNA at 37°C for 3h
RNase residue	Degradation of 293-RNA does not exceed 10%	5U enzyme and 293-RNA. Incubate at 37°C for 1h
Bacterial endotoxin content	≤10 EU/mg	Chinese Pharmacopoeia 2020 Edition Fourth Gel Limit Test Method (General Rule 1143)
Host DNA residue	≤100 pg/mg	Fluorescence quantitative PCR
Host protein residue	≤50 ppm	Chinese Pharmacopoeia 2020 Edition Part IV Method for the Determination of Bacterial Protein Residues (General Rule 3412)
Mycoplasma	Feminine	Mycoplasma detection kit
Heavy metal residue	≤10 ppm	Chinese Pharmacopoeia 2020 Edition Fourth Heavy Metal Inspection Method (General Principle 0821)

Follow the following specifications for production

1. ISO 9001:2015, certified facility.

2. "GMP Appendix-Cell Therapy Products" State Drug Administration.

3. "General Introduction to Human Gene Therapy-Chinese Pharmacopoeia 2020" National Pharmacopoeia Commission.

4. USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products are used as excipients in cell therapy, gene therapy and tissue engineering products.

5. USP Chapter <92>, Growth Factors and Cytokines Used in Cell Therapy Manufacturing Cytokines and growth factors used in the production of cell therapy products.

6.Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products.

Product Usage

1. Used for RNA 3´ end labeling.

2. Add Poly(A) tail to RNA for cloning or affinity purification. For example, adding Poly (A) to miRNA provides an oligo-dT primer binding site for cDNA synthesis.

3. Improve the translation efficiency of RNA in eukaryotic cells.

Preservation system

20mM Tris-HCl; 300mM NaCl; 1mM DTT; 1mM EDTA; 0.1% TritonX-100; 50% (v/v) Glycerol, pH 7.5.

Applications

The intact mRNA expresses GFP protein in the cell, the capping enzyme is compared with the cap analog

Precautions

1. Thermal inactivation conditions: 65°C, 20 min.

2. The enzyme can only use RNA as a substrate.

3. The enzyme adds AMP to the 3´ end of RNA with high selectivity, and does not add the same length of Poly (A) to all RNA molecules.

4. The enzyme uses M-MuLV reverse transcriptase reaction buffer, which can also be used for reaction.

5. The enzyme requires divalent cations such as Mg2+ to be active.

6. The length of RNA plus A tail is affected by the amount of enzyme, ATP, reaction time and other factors. The amount of A required for different experiments will be different. The length of A can be adjusted by reducing the reaction time. The enzyme is at 37°C. About 30 A bases can be added in 30 minutes of reaction, and about 100 A bases can be added in 1 hour.

7. EDTA inhibits the enzyme activity. If the reaction is stopped, it can be purified directly by adding EDTA to a final concentration of 10mM.

Specifications

Product Name

E. coli Poly(A) Polymerase

Grado

Pharmaceutical grade, PharmPure™

Especificaciones y pureza

pharmaceutical grade, PharmPure™

Tipo de molécula

Enzyme

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at -20°C

Enviado en

Ice chest + Ice pads

Definición de unidad

在 37℃条件下，10 min 内将1 nmol AMP 掺入到 RNA 所需的酶量定义为 1 个活性单位（U）