IL-21 production in prokaryotic hosts

Abstract

The expression vectors and methods using an E. coli expression system for the large scale production of IL-21 are described. The vectors utilize the IL-21 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA secondary structure for translation in E. coli. Using the expression vectors, the IL-21 gene was produced in E. coli to a level of greater than 1 g / L in fed batch fermentation. Also included are OmpT deficient E. coli strains transformed with an IL-21 expression vector.

Description

Background of the invention

[0001] More genes can be identified and obtained from the genomes of humans and other organisms, increasing the need for efficient expression and purification of recombinant proteins. Protein expression in bacteria is by far the most widely used way to produce cloned proteins. Prokaryotic cells are preferred over eukaryotic cells for expression for a number of reasons. For example, it is much easier to grow bacteria than eukaryotic cells. More particularly, Escherichia coli has become an extremely useful expression host for protein production due to the availability of a large number of sophisticated molecular genetic tools and thousands of mutants. But producing large quantities of functional proteins in E. coli, especially those of eukaryotic origin, is often difficult.

[0002] IL-21 (formerly named Zalphal 1 ligand) is a member of the IL-2 cytokine family, which also includes IL-4, IL-7, IL-9, IL13, and IL-15. Proteins of this family have be...

Examples

Embodiment 1

[0116] Example 1 Construction of expression vector pTAP237

[0117] Plasmid pTAP237 was obtained by inserting a PCR-generated linker at the SmaI site of pTAP186 by homologous recombination. Plasmid pTAP186 was derived from plasmid pRS316 (a shuttle vector for Saccharomyces cerevisiae) and plasmid pMAL-c2 (an E. coli expression plasmid derived from pKK223-2 and containing tac promoter and rrnb terminator). Plasmid pTAP186 contains a kanamycin resistance gene in which the SmaI site is disrupted and has the yeast ARS-CEN6 and URA3 sequences surrounded by sites NotI and SfiI, making these two sequences cleavable by NotI down. The PCR-generated linker replaced the expression coupling sequence in pTAP186 with a synthetic RBSII sequence. The sequence was prepared using 100 pmol of various oligonucleotides zc29,740 and zc29,741 (respectively shown in SEQ ID NO: 3 and 4) and approximately 5 pmol of various oligonucleotides (respectively shown in SEQ ID NO : 5 and 6). The oligonucle...

Embodiment 2

[0119] Construction of pTAP252

[0120] The coding sequence of human IL-21 (as shown in SEQ ID NO: 1) was amplified by PCR, using the CD3+cDNA library as a template, and oligonucleotide primers zc29,084 and zc22,127 (identified by SEQ ID NO : 7 and 8). In order to optimize the translation process in E. coli, primer zc29,084 (shown in SEQ ID NO: 7) was used to add an ATG initiation codon to the 5' end of the IL-21 coding sequence. The resulting gene sequence encodes mature L-21 with an extra methionine at the N-terminus (IL-21met). The final PCR product was inserted into the expression vector pTAP237 (described in Example 1) by yeast homologous recombination (Raymond et al., Biotechniques.26 (1): 134-8, 140-1, 1999; U.S. Patent No. 6,027,442 , cited here as a reference). The expression construct pTAP252 was extracted from yeast and transformed into competent E. coli MC1061. Kanamycin-resistant clones were identified by colony PCR. Positive clones were confirmed by sequenci...

Embodiment 3

[0122] codon optimization

[0123]Introduction of pTAP252 to express human IL-21met in E. coli strain E104 yielded approximately 2-5% of total cellular protein. Examination of the codons used in the IL-21 coding sequence showed an excess of codons rarely used in E. coli with a CAI value of 0.181. CAI is a statistical measure of synonymous codon preference and can be used to predict the level of protein expression (Sharp et al., Nucleic Acids Res. 15(3):1281-95, 1987). Genes encoding highly expressed proteins often have high CAI values ​​(>0.6), while genes encoded by genes with low CAI values ​​(<0.2) generally express lower efficiency. This suggests a reason for the low production of IL-21 in E. coli. In addition, rare codons are clustered in the second half of the message chain, making translation pauses, premature termination of translation, and high probability of amino acid misincorporation (Kane JF.Curr.Opin.Biotechnol.6(5):494- 500, 1995).

[0124] It has been shown...

Background of the invention

[0001] More genes can be identified and obtained from the genomes of humans and other organisms, increasing the need for efficient expression and purification of recombinant proteins. Protein expression in bacteria is by far the most widely used way to produce cloned proteins. Prokaryotic cells are preferred over eukaryotic cells for expression for a number of reasons. For example, it is much easier to grow bacteria than eukaryotic cells. More particularly, Escherichia coli has become an extremely useful expression host for protein production due to the availability of a large number of sophisticated molecular genetic tools and thousands of mutants. But producing large quantities of functional proteins in E. coli, especially those of eukaryotic origin, is often difficult.

[0002] IL-21 (formerly named Zalphal 1 ligand) is a member of the IL-2 cytokine family, which also includes IL-4, IL-7, IL-9, IL13, and IL-15. Proteins of this family have be...

Examples

Embodiment 1

[0116] Example 1 Construction of expression vector pTAP237

[0117] Plasmid pTAP237 was obtained by inserting a PCR-generated linker at the SmaI site of pTAP186 by homologous recombination. Plasmid pTAP186 was derived from plasmid pRS316 (a shuttle vector for Saccharomyces cerevisiae) and plasmid pMAL-c2 (an E. coli expression plasmid derived from pKK223-2 and containing tac promoter and rrnb terminator). Plasmid pTAP186 contains a kanamycin resistance gene in which the SmaI site is disrupted and has the yeast ARS-CEN6 and URA3 sequences surrounded by sites NotI and SfiI, making these two sequences cleavable by NotI down. The PCR-generated linker replaced the expression coupling sequence in pTAP186 with a synthetic RBSII sequence. The sequence was prepared using 100 pmol of various oligonucleotides zc29,740 and zc29,741 (respectively shown in SEQ ID NO: 3 and 4) and approximately 5 pmol of various oligonucleotides (respectively shown in SEQ ID NO : 5 and 6). The oligonucle...

Embodiment 2

[0119] Construction of pTAP252

[0120] The coding sequence of human IL-21 (as shown in SEQ ID NO: 1) was amplified by PCR, using the CD3+cDNA library as a template, and oligonucleotide primers zc29,084 and zc22,127 (identified by SEQ ID NO : 7 and 8). In order to optimize the translation process in E. coli, primer zc29,084 (shown in SEQ ID NO: 7) was used to add an ATG initiation codon to the 5' end of the IL-21 coding sequence. The resulting gene sequence encodes mature L-21 with an extra methionine at the N-terminus (IL-21met). The final PCR product was inserted into the expression vector pTAP237 (described in Example 1) by yeast homologous recombination (Raymond et al., Biotechniques.26 (1): 134-8, 140-1, 1999; U.S. Patent No. 6,027,442 , cited here as a reference). The expression construct pTAP252 was extracted from yeast and transformed into competent E. coli MC1061. Kanamycin-resistant clones were identified by colony PCR. Positive clones were confirmed by sequenci...

Embodiment 3

[0122] codon optimization

[0123]Introduction of pTAP252 to express human IL-21met in E. coli strain E104 yielded approximately 2-5% of total cellular protein. Examination of the codons used in the IL-21 coding sequence showed an excess of codons rarely used in E. coli with a CAI value of 0.181. CAI is a statistical measure of synonymous codon preference and can be used to predict the level of protein expression (Sharp et al., Nucleic Acids Res. 15(3):1281-95, 1987). Genes encoding highly expressed proteins often have high CAI values ​​(>0.6), while genes encoded by genes with low CAI values ​​(<0.2) generally express lower efficiency. This suggests a reason for the low production of IL-21 in E. coli. In addition, rare codons are clustered in the second half of the message chain, making translation pauses, premature termination of translation, and high probability of amino acid misincorporation (Kane JF.Curr.Opin.Biotechnol.6(5):494- 500, 1995).

[0124] It has been shown...

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