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Production and purification of il-29

a technology of il-29 and purification, which is applied in the field of production and purification of il-29, can solve the problems of difficult high-level production of functional proteins in i>e. coli/i>., especially those from eukaryotic sources, and achieves the effect of improving the quality of i>e. coli/i>

Inactive Publication Date: 2008-04-24
ZYMOGENETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high-level production of functional proteins in E. coli., especially those from eukaryotic sources has often been difficult.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of expression vector, pTAP395

[0136]The backbone used to construct the E. coli expression vector for IL-29 C172S d2-7 (SEQ ID NO:5) was pTAP395. pTAP395 contained the srp promoter, two transcriptional terminators, rrnB T1 and rrnB T2, kanamycin resistance gene, origin of replication, URA3 selection marker and the ARS-CENS6 locus for plasmid replication in yeast. pTAP395 was generated from pTAP238 but has a different translational enhancer from pTAP238. pTAP395 had the translational enhancer known as chan2 or zymo2 (SEQ ID NO:13). pTAP395 was constructed using oligos zc42188 (SEQ ID NO:14), zc42187 (SEQ ID NO:15), zc42194 (SEQ ID NO:16), and zc29741(SEQ ID NO:17) by implementing an overlap-PCR strategy. The ends of the PCR fragment were homologous to pTAP395. The central region between the XbaI and SmaI sites contained the zymo2 ((SEQ ID NO:13)) translational enhancer. The PCR reagent concentrations were as follows: 1 μM of zc42188 (SEQ ID NO:14) and zc29741 (SEQ ID NO:17...

example 2

Construction of codon optimized IL-29 gene

[0137]The IL-29 coding sequence with codon optimized for translation in E. coli was constructed from ten overlapping oligonucleotides (Oligo number: zc44,559 (SEQ ID NO:18), zc44,566 (SEQ ID NO:19), zc44,565 (SEQ ID NO:20), zc44,562 (SEQ ID NO:21), zc44,563 (SEQ ID NO:22), zc44,560 (SEQ ID NO:23), zc44,561 (SEQ ID NO:24), zc44,564 (SEQ ID NO:25), zc44,557 (SEQ ID NO:26) and zc44,558 (SEQ ID NO:27). Primer extension followed by PCR amplification produced a full-length, optimized IL-29 gene. The final PCR product was inserted into the cloning vector, pCR-Blunt II TOPO by ligation. The ligation mix was transformed into competent E. coli TOP10. Kanamycin resistant clones were screened by colony PCR. A positive clone was verified by DNA sequencing.

example 3

Construction of expression vector pCHAN15

[0138]The strategy used to generate the IL-29 C172S (SEQ ID NO:1) mutant is based on the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.). Primers were designed to introduce the C172S mutation according to the manufacturer's suggestions. The primers were designated zc44,340 (SEQ ID NO:28) and zc44,341 (SEQ ID NO:29). PCR was performed to generate the IL-29 C172S mutant according to instructions provided with the QuikChange Mutagenesis Kit. Five identical 50 μL reactions were set-up. Each reaction contained 2.5 μL of pSDH175 (expression construct with the optimized IL-29 gene sequence) as template. The PCR cocktail contained the reagents: 30 μL 10×PCR buffer, 125 ng (27.42 μL) zc44,340 (SEQ ID NO:28). 125 ng (9.18 μL) zc44,341 (SEQ ID NO:29), 6 μL DNTP, 6 μL Pfu Turbo polymerase (Strategene), and 206.4 μL water. Each reaction received 47.5 μL of the cocktail. The PCR conditions were as follows: 1 cycle of 95° C. for 30 ...

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Abstract

The expression vectors and methods using an E. Coli expression system for the large scale production of IL-29 are described. The vectors utilize the IL-29 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA secondary structure for translation in E. coli. Also included are methods of producing, purifying and pegylating an IL-29 polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application claims the benefit of U.S. Patent Application Ser. No. 60 / 723,544, filed Oct. 4, 2005, which is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]The increased availability and identification of genes from human and other genomes has led to an increased need for efficient expression and purification of recombinant proteins. The expression of proteins in bacteria is by far the most widely used approach for the production of cloned genes. For many reasons, expression in bacteria is preferred to expression in eukaryotic cells. For example, bacteria are much easier to grow than eukaryotic cells. More specifically, the availability of a wealth of sophisticated molecular genetic tools and thousands of mutants make E. Coli, as an expression host, extremely useful for protein production. However, the high-level production of functional proteins in E. coli., especially those from eukaryotic s...

Claims

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Application Information

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IPC IPC(8): C07K14/54C07K1/18C12P21/00
CPCC12P21/02C07K14/54
Inventor ZAMOST, BRUCE L.LEE, GEOFFREY F.DEDINSKY, ROBERT M.
Owner ZYMOGENETICS INC
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