摘要:
The invention relates to a process for the recombinant production of a heterologous polypeptide of interest by cultivating a bacterial host cell transformed with an expression vector comprising a nucleic acid molecule encoding a fusion polypeptide wherein (a) the amino-proximal fusion partner is an autoprotease Npro comprising the replacement(s) by glutamic acid of one or more cysteines at positions corresponding to the positions 112, 134, and 138 of the autoprotease Npro of classical swine fever virus and (b) the carboxyl-proximal fusion partner is an heterologous polypeptide of interest fused to the autoprotease Npro so that it is capable of being cleaved from the fusion polypeptide by autoprotease Npro autoproteolytic activity, said process comprising (i) cultivating the transformed host cell under conditions permitting the expression of the fusion polypeptide and the formation of corresponding cytoplasmic inclusion bodies, (ii) isolating the inclusion bodies from the host cell, (iii) solubilizing the isolated inclusion bodies, (iv) inducing autoproteolytic cleavage of the heterologous polypeptide of interest from the fusion polypeptide, and (v) isolating the cleaved heterologous polypeptide of interest.
摘要:
The invention relates to a process for the recombinant production of a heterologous polypeptide of interest, comprising, (i) cultivation of a bacterial host cell which is transformed with an expression vector which comprises a nucleic acid molecule which codes for a fusion polypeptide, the fusion polypeptide comprising a derivative of an autoprotease Npro of Pestivirus, wherein at least one cysteine residue of the naturally occuring autoprotease Npro of Pestivirus is replaced by another amino acid residue, and a second polypeptide which is connected to the first polypeptide at the C-terminus of the first polypeptide in a manner such, that the second polypeptide is capable of being cleaved from the fusion polypeptide by the autoproteolytic activity of the first polypeptide, said second polypeptide being a heterologous polypeptide, wherein cultivation occurs under conditions which cause expression of the fusion polypeptide and formation of corresponding cytoplasmic inclusion bodies, (ii) isolation of the inclusion bodies from the host cell, (iii) solubilization of the isolated inclusion bodies, (iv) induction of autoproteolytic cleavage of the heterologous polypeptide of interest from the fusion polypeptide, and (v) isolation of the cleaved heterologous polypeptide of interest.
摘要:
Disclosed is an affinity matrix comprising a solid phase and an affinity ligand comprising peptide bonds coupled to this solid phase, wherein the affinity ligand comprising peptide bond is selected from the following group of ligands: a) peptides comprising the formula X1X2X3X4, wherein X1 to X4 are amino acid residues and at least two of X1 to X4 is W, Y or F; b) peptides comprising the formula X5X6X7X8, wherein X5 to X8 are amino acid residues, at least one of X5 to X8 is W, and at least one of X5 to X8 is E or D; and c) poly-amino acids consisting of an amino acid monomer of the group consisting of R, K, E and D and an amino acid monomer of the group consisting of Y, F and W, preferably poly-KY, poly-KF, poly-KW, poly-RY, poly-RF, poly-RW, poly-EY, poly-DY, poly-EF, poly-EW, poly-DF and poly-DW, with the proviso that the peptides according to a) and b) have a maximum length of 35 amino acid residues and that the poly-amino acids according to c) have a minimum length of 20 amino acid residues.
摘要:
Disclosed is an affinity matrix comprising a solid phase and an affinity ligand comprising peptide bonds coupled to this solid phase, wherein the affinity ligand comprising peptide bond is selected from the following group of ligands: a) peptides comprising the formula X1X2X3X4, wherein X1 to X4 are amino acid residues and at least two of X1 to X4 is W, Y or F; b) peptides comprising the formula X5X6X7X8, wherein X5 to X8 are amino acid residues, at least one of X5 to X8 is W, and at least one of X5 to X8 is E or D; and c) poly-amino acids consisting of an amino acid monomer of the group consisting of R, K, E and D and an amino acid monomer of the group consisting of Y, F and W, preferably poly-KY, poly-KF, poly-KW, poly-RY, poly-RF, poly-RW, poly-EY, poly-DY, poly-EF, poly-EW, poly-DF and poly-DW, with the proviso that the peptides according to a) and b) have a maximum length of 35 amino acid residues and that the poly-amino acids according to c) have a minimum length of 20 amino acid residues.
摘要:
An scFv peptide comprising a VH domain and a VL domain linked by an amino acid spacer is disclosed. The VH domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 64. The VL domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 66. The scFv peptide also comprises the substitution or deletion of an amino acid in the VH domain at the position corresponding to C28.
摘要翻译:公开了包含由VH结构域和通过氨基酸间隔物连接的VL结构域的scFv肽。 VH结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 VL结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 scFv肽还包括在对应于C28的位置上的VH结构域中的氨基酸的取代或缺失。
摘要:
An scFv peptide comprising a VH domain and a VL domain linked by an amino acid spacer is disclosed. The VH domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 64. The VL domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 66. The scFv peptide also comprises the substitution or deletion of an amino acid in the VH domain at the position corresponding to C28.
摘要翻译:公开了包含由VH结构域和通过氨基酸间隔物连接的VL结构域的scFv肽。 VH结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 VL结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 scFv肽还包括在对应于C28的位置上的VH结构域中的氨基酸的取代或缺失。
摘要:
An scFv peptide comprising a VH domain and a VL domain linked by an amino acid spacer is disclosed. The VH domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 64. The VL domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 66. The scFv peptide comprises an additional feature selected from (a) to (e) and combinations thereof. (a) A substitution or deletion of an amino acid in the VH domain at a position corresponding to that selected from the group consisting of: I29, H68, N85, C97 and combinations thereof. (b) A substitution or deletion of an amino acid in the VL domain at a position corresponding to that selected from the group consisting of: V2, V3, F10, F14, A39, N76 and combinations thereof. (c) The amino acid spacer comprises the sequence (GGGGS)n wherein n is between 4 and 6. (d) The VH domain further comprises an N-terminal pelB signal sequence comprising the sequence of SEQ ID NO. 68 or a sequence having at least 80% sequence identity thereto. (e) The VL domain is located at the N-terminal end of the VH domain. (f) The substitution of the cysteine residue in the VH domain corresponding to position 28 with a serine residue.
摘要翻译:公开了包含由VH结构域和通过氨基酸间隔物连接的VL结构域的scFv肽。 VH结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 VL结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 scFv肽包含选自(a)至(e)的附加特征及其组合。 (a)在与选自:I29,H68,N85,C97及其组合的对应位置的VH结构域中的氨基酸的取代或缺失。 (b)在对应于选自V2,V3,F10,F14,A39,N76及其组合的对应位置的VL结构域中的氨基酸的取代或缺失。 (c)氨基酸间隔物包含其中n在4和6之间的序列(GGGGS)n。(d)VH结构域还包含N末端pelB信号序列,其包含SEQ ID NO: 68或与其具有至少80%序列同一性的序列。 (e)VL结构域位于VH结构域的N末端。 (f)用丝氨酸残基取代对应于位置28的VH结构域中的半胱氨酸残基。
摘要:
An scFv peptide comprising a VH domain and a VL domain linked by an amino acid spacer is disclosed. The VH domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 64. The VL domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 66. The scFv peptide also comprises the substitution or deletion of an amino acid in the VH domain at the position corresponding to C28.
摘要翻译:公开了包含由VH结构域和通过氨基酸间隔物连接的VL结构域的scFv肽。 VH结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 VL结构域包含与SEQ ID NO:1的序列具有至少80%的序列同一性的序列。 scFv肽还包括在对应于C28的位置上的VH结构域中的氨基酸的取代或缺失。