Extended recombinant polypeptides and compositions comprising same
By connecting the extended recombinant polypeptide XTEN with a bioactive protein to form a fusion protein, the instability and production complexity of bioactive proteins in aqueous solutions are solved, the half-life is extended, the solubility is improved, and the biological activity is enhanced, and production is simplified. process.
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Embodiment 1
[0509] Embodiment 1: Construction of XTEN_AD36 motif segment
[0510] The following example describes the construction of a codon-optimized gene collection encoding a 36 amino acid motif. In the first step, a stuffer vector pCW0359 was constructed based on the pET vector, which contained the T7 promoter. pCW0359 encodes the cellulose binding domain (CBD) and the TEV isoflurane recognition site followed by a stuffer sequence. The stuffer sequence is flanked by BsaI, BbsI and KpnI sites. BsaI and BbsI sites were inserted such that they produced compatible overhangs after digestion. This stuffer sequence is followed by a truncated version 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 the stuffer segment and the resulting vector fragments were isolated by agarose gel purification. The sequence wa...
Embodiment 2
[0524] Example 2: Construction of the XTEN_AE36 segment
[0525] A codon library encoding XTEN sequences with a length of 36 amino acids was constructed. Name the XTEN sequence XTEN_AE36. Its segment has the amino acid sequence [X] 3 , wherein X is a 12mer peptide, the sequence is: GSPAGSPTSTEE (SEQ ID NO: 359), GSEPATSGSE TP (SEQ ID NO: 360), GTSESA TPESGP (SEQ ID NO: 361) or GTTEPSEGSAP (SEQ ID NO: 362). Inserts were obtained by annealing the following pairs of phosphorylated synthetic oligonucleotides:
[0526] AE1 for: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA (SEQ ID NO: 363)
[0527] AE1rev: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT (SEQ ID NO: 364)
[0528] AE2 for: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC (SEQ ID NO: 365)
[0529] AE2rev: ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT (SEQ ID NO: 366)
[0530] AE3 for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC (SEQ ID NO: 367)
[0531] AE3rev: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT (SEQ ID NO: 368)
[0532] AE4 for: AGGTACYTCTACYGAACCKTCYGAR...
Embodiment 3
[0540] Example 3: Construction of the XTEN_AF36 segment
[0541] A codon library encoding sequences 36 amino acids in length was constructed. Name the sequence XTEN_AF36. Its segment has the amino acid sequence [X] 3 , wherein X is a 12mer peptide, the sequence is: GSTSESPSGTAP (SEQ ID NO: 447), GTSTPESGSASP (SEQ ID NO: 448), GTSPSGESSTAP (SEQ ID NO: 449) or GSTSSTAESPGP (SEQ ID NO: 450). Inserts were obtained by annealing the following pairs of phosphorylated synthetic oligonucleotides:
[0542] AF1 for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC (SEQ ID NO: 451)
[0543] AF1rev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA (SEQ ID NO: 452)
[0544] AF2 for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC (SEQ ID NO: 453)
[0545] AF2rev: ACCTGGAGAWGCRGARCCGCTTTTCMGGRGTAGARGT (SEQ ID NO: 454)
[0546] AF3 for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC (SEQ ID NO: 455)
[0547] AF3rev: ACCTGGWGCRGTAGAAAGATTCRCCGCTMGGRGARGT (SEQ ID NO: 456)
[0548] AF4 for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC (SEQ...
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