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Recombinantly expressed carboxypeptidase b and purification thereof

A carboxypeptidase, precarboxypeptidase technology, used in the production and further processing of inactive precursors, the field of protein activation, and can solve the problem that there is no known specific method to distinguish activated carboxypeptidase B from inactive forms, etc. question

Active Publication Date: 2005-06-08
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] To the best of the present inventors' knowledge, there is currently no known specific method for distinguishing activated carboxypeptidase B from the inactive form

Method used

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  • Recombinantly expressed carboxypeptidase b and purification thereof
  • Recombinantly expressed carboxypeptidase b and purification thereof
  • Recombinantly expressed carboxypeptidase b and purification thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 9

[0094] Example 9 shows that after the first purification step as in Example 7, the collected pool contains almost equal amounts of products with and without C-terminal tyrosine product of. Terminal tyrosine residues are gradually removed during processing. After the second chromatographic purification step, the C-terminal tyrosine was almost completely deleted for most proteinaceous materials. A possible but unproven explanation is therefore that the host organism used to express and secrete the product produces the protein with carboxypeptidase Y activity. Carboxypeptidase Y is known from Saccharomyces cerevisiae with the ability to cleave the C-terminal amino acid with broad specificity. Although carboxypeptidase Y is known to be a vacuolar enzyme (Kato, M. et al., Eur. J. Biochem 270 (2003) 4587-4593), lysed yeast cells can significantly increase carboxypeptidase in the fermentation medium. Y activity. However, since removal of the C-terminal tyrosine did not alter the ...

Embodiment 1

[0110] Synthesis of genes encoding precursor proteins

[0111] DNA manipulation techniques were carried out according to standard protocols (Sambrook, Fritsh & Maniatis, Molecular Cloning, A Laboratory Manual, 3 rd Edition, CSHL Press, 2001). Reagents used in molecular biology experiments were used according to the manufacturer's recommendations.

[0112] A nucleotide sequence encoding artificial preprocarboxypeptidase B gene as shown in SEQ ID NO:3 was re-synthesized. Several parts of the nucleotide sequence were synthesized as 24 single-stranded DNA oligonucleotides ranging in length from 54 to 90 nucleotides. The single-stranded oligonucleotide represents an alternating overlapping portion of the leading and lagging strands of SEQ ID NO:3. Each oligonucleotide was designed such that its 5' and 3' ends overlapped adjacent oligonucleotides. As an exception, oligonucleotides representing the 5' and 3' ends of SEQ ID NO: 3 only overlap the 5' and 3' ends of their adjacent ...

Embodiment 2

[0118] Vector construction, transformation, expression

[0119] For the construction of the expression vector, transformation, expression of the precursor protein and secretion of histidine-tagged procarboxypeptidase B in the growth medium, the "Pichia Yeast Expression Kit" model M 011102 25 described in the Invitrogen brochure was used -0043, "pPICZ A, B, and C" model D 110801 25-0148, "pPECZα A, B, and C" model E 01030225-0150, and "pPIC9K" model E030402 25-0106 to perform experiments. Reference is also made to other vectors, yeast strains and media mentioned herein. At the same time, such as Sambrook, Fritsh&Maniatis, Molecular Cloning, A Laboratory Manual, 3 rd Edition, CSHL Press, 2001 describes the basic molecular biology methods.

[0120] As a result, a number of vectors were obtained, each of which contained an expression cassette capable of expressing the precursor protein shown in SEQ ID NO:4 among the methylotrophic yeast strains. A preferred yeast strain is a P...

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Abstract

The invention provides a method to produce a protein with carboxypeptidase B activity from a pro-carboxypeptidase B zymogen, derived from a non-animal host organism. Carboxypeptidase B is activated from the zymogen using non-denaturing conditions. Particularly, the activation is performed under conditions that avoid unwanted non-covalent binding of the propeptide to the activated carboxypeptidase B enzyme.

Description

technical field [0001] The present invention relates to the field of biotechnology. More particularly, the present invention relates to the production and further processing of an enzymatically inactive precursor of a protein having carboxypeptidase B activity. One aspect of the invention relates to the activation and concomitant purification of a protein having carboxypeptidase B activity. Background technique [0002] Carboxypeptidase is an enzyme that hydrolyzes the C-terminal peptide bond. The carboxypeptidase family includes metallo, serine, and cysteine ​​carboxypeptidases. Depending on their substrate specificity, these enzymes are called carboxypeptidase A (cleaves aliphatic residues) or carboxypeptidase B (cleaves basic amino acid residues). Carboxypeptidase B (EC3.4.17.2) is an enzyme that catalyzes the hydrolysis of the basic amino acids, lysine, arginine, and ornithine, from the C-terminal position of polypeptides: [0003] <chemistry num="001"> <c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/48
CPCC07K2319/02C07K2319/21C12N9/48
Inventor S·格拉瑟F·盖佩尔T·柯施鲍姆B·雷克塞尔J·-P·塔尔霍菲R·米勒C·吉塞尔H·埃克施泰因E·沃尔夫
Owner F HOFFMANN LA ROCHE & CO AG
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