Extended recombinant polypeptides and compositions comprising same

A technology for extending recombinant polypeptides and summaries, applied in the field of copyright, can solve problems such as expensive, additional production steps, complex purification operations, etc.

Active Publication Date: 2012-02-08
阿穆尼克斯制药公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a time-consuming, inefficient, and expensive process that requir

Method used

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  • Extended recombinant polypeptides and compositions comprising same
  • Extended recombinant polypeptides and compositions comprising same
  • Extended recombinant polypeptides and compositions comprising same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0458] Example 1: Construction of XTEN_AD36 motif segment

[0459] The following examples describe the construction of a codon-optimized gene set encoding a 36 amino acid motif. In the first step, a stuffer vector pCW0359 is constructed based on the pET vector, which contains the T7 promoter. pCW0359 encodes a cellulose binding domain (CBD) and a TEV isoflurane recognition site, followed by a stuffer sequence. The stuffer sequence is flanked by BsaI, BbsI and KpnI sites. Insert the BsaI and BbsI sites so that they produce compatible overhangs after digestion. The stuffer sequence is followed by a truncated form of the GFP gene and a His tag. The stuffer sequence contains a stop codon, so E. coli cells carrying the stuffer plasmid pCW0359 form non-fluorescent colonies. The stuffer vector pCW0359 was digested with BsaI and KpnI to remove stuffer segments, and the resulting vector fragments were separated by agarose gel purification. The sequence was named XTEN_AD36, reflecting...

Embodiment 2

[0473] Example 2: Construction of XTEN_AE36 segment

[0474] A codon library encoding XTEN sequences of 36 amino acids in length was constructed. The XTEN sequence was named XTEN_AE36. Its segment has an amino acid sequence [X] 3 , Where X is a 12mer peptide, and the sequence is: GSPAGSPTSTEE (SEQ ID NO: 359), GSEPATSGSE TP (SEQ ID NO: 360), GTSESA TPESGP (SEQ ID NO: 361) or GTSTEPSEGSAP (SEQ ID NO: 362). The insert is obtained by annealing the following phosphorylated synthetic oligonucleotide pairs:

[0475] AE1for: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA (SEQ ID NO: 363)

[0476] AE1rev: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT (SEQ ID NO: 364)

[0477] AE2for: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC (SEQ ID NO: 365)

[0478] AE2rev: ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT (SEQ ID NO: 366)

[0479] AE3for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC (SEQ ID NO: 367)

[0480] AE3rev: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT (SEQ ID NO: 368)

[0481] AE4for: AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC (SEQ ID NO: 36...

Embodiment 3

[0490] Example 3: Construction of XTEN_AF36 segment

[0491] A codon library encoding a sequence of 36 amino acids in length was constructed. This sequence was named XTEN_AF36. Its segment has an amino acid sequence [X] 3 , Where X is a 12mer peptide, and the sequence is: GSTSESPSGTAP (SEQ ID NO: 447), GTSTPESGSASP (SEQ ID NO: 448), GTSPSGESSTAP (SEQ ID NO: 449) or GSTSSTAESPGP (SEQ ID NO: 450). The insert is obtained by annealing the following phosphorylated synthetic oligonucleotide pairs:

[0492] AF1for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC (SEQ ID NO: 451)

[0493] AF1rev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA (SEQ ID NO: 452)

[0494] AF2for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC (SEQ ID NO:453)

[0495] AF2rev: ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT (SEQ ID NO: 454)

[0496] AF3for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC (SEQ ID NO: 455)

[0497] AF3rev: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT (SEQ ID NO: 456)

[0498] AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC (SEQ ID NO: 457)

[0499] ...

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Abstract

The present invention relates to compositions comprising biologically active proteins linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of using such compositions in treatment of glucose- related diseases, metabolic diseases, coagulation disorders, and growth hormone-related disorders and conditions.

Description

[0001] Statement on Federally Funded Research [0002] This invention was completed with government support under the SBIR Fund 2R44GM079873-02 funded by the National Institutes of Health. The government has certain rights in this invention. [0003] Cross references to related applications [0004] This application requires the U.S. Provisional Application No. 61 / 149,669 filed on February 3, 2009, 61 / 268,193 filed on June 8, 2009, 61 / 185,112 filed on June 8, 2009, August 24, 2009 61 / 236,493 filed on August 25, 2009, 61 / 236,836 filed on August 25, 2009, 61 / 243,707 filed on September 18, 2009, 61 / 245,490 filed on September 24, 2009, and filed on November 10, 2009 The priority of 61 / 280,955, 61 / 280,956 filed on November 10, 2009, and 61 / 281,109 filed on November 12, 2009, the above applications are all pending, and are incorporated herein by reference in their entirety. Background of the invention [0005] Biologically active proteins, including those used as therapeutic agents, are g...

Claims

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Application Information

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IPC IPC(8): C07K1/00C07K14/00C07K17/00
CPCC07K14/001C07K2319/31C07K14/61C07K14/745C07K2319/35C12N9/6437C12N9/644C07K14/605A61P1/00A61P1/04A61P1/14A61P19/02A61P21/00A61P21/04A61P25/00A61P25/02A61P27/02A61P29/00A61P3/04A61P31/18A61P3/06A61P3/08A61P43/00A61P5/00A61P5/06A61P5/10A61P5/22A61P5/48A61P5/50A61P7/00A61P7/02A61P7/04A61P7/06A61P9/00A61P9/10A61P9/12A61P9/14A61P3/10A61K38/16C07K1/00C07K14/00C07K14/435C07K19/00C07K14/47C07K14/545
Inventor V·舍伦伯格J·希尔弗曼王家威B·斯平克W·P·斯特默N·吉辛W·托J·L·克兰德
Owner 阿穆尼克斯制药公司
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