Process for production of insulin and insulin analogues
Inventors
KRISHNAN, Archana • SONAR, Sanjay • THAPPA, Damodar
Assignees
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Publication Number
US-10000544-B2
Publication Date
2018-06-19
Expiration Date
Abstract
A process for production of insulin or insulin analogs by expression of Insulin or Insulin analogs through an expression vector in a host cell is provided. The expression vector includes a leader peptide of SEQ ID NO 3; a nucleotide sequence encoding an affinity tag linked to C-terminal end or N terminal end of nucleotide sequence of the leader peptide; and a nucleotide sequence encoding for a cleavage site ligated to nucleotide sequence of the leader peptide through nucleotide sequence encoding the affinity tag.
Core Innovation
The invention relates to a process for production of an insulin or an insulin analogue in bacteria through an expression vector. The expression vector comprises a first nucleotide sequence encoding a leader peptide of SEQ ID NO:3, a second nucleotide sequence encoding an affinity tag, and a third nucleotide sequence encoding a cleavage site that is ligated to the leader peptide or to the affinity tag. In this way, the leader peptide and the affinity tag are arranged in a defined manner on the expression product in bacteria prior to cleavage.
The expression-vector design further specifies that the affinity tag is linked to either a C-terminal end or an N-terminal end of the leader peptide, while the cleavage-site nucleotide sequence is ligated to the leader peptide or to the affinity tag. The disclosed leader peptide has N-terminal Met followed by Gly and includes sequence properties intended to support production in bacteria. The document also discloses vector sequence features and arrangement, including a multiple cloning site (MCS), a ribosome binding site (RBS), a promoter or operator sequence, and an antibiotic selection marker.
After expression, the document describes downstream conversion to mature insulin or insulin analogues by processing that includes refolding and proteolysis, including digestion steps using proteases. The conversion is exemplified for human insulin and insulin analogues including Aspart and Lispro, with evidence provided by MALDI-TOF and SDS-PAGE to support generation consistent with the claimed single construct digestion approach from proinsulin fusion constructs.
Claims Coverage
The document includes three independent process claims covering production of an insulin or an insulin analogue by bacterial expression using an expression vector with a leader peptide of SEQ ID NO:3, an affinity tag linked to the leader peptide, and a cleavage-site or restriction enzyme site arrangement enabling expression-product processing. The claims further define vector architecture elements and specify particular sequence identity content (SEQ ID NO:1) and optional narrowing to specific bacterial host components.
Bacterial expression vector with SEQ ID NO:3 leader, affinity tag, and ligated cleavage site
Expressing an insulin or an insulin analogue through an expression vector in bacteria, wherein the expression vector comprises a first nucleotide sequence encoding a leader peptide of SEQ ID NO:3; a second nucleotide sequence encoding an affinity tag linked to a C-terminal end or N-terminal end of the leader peptide; and a third nucleotide sequence encoding a cleavage site ligated to the first nucleotide sequence encoding the leader peptide or to the second nucleotide sequence encoding the affinity tag.
Bacterial expression vector with cleavage site or restriction enzyme site plus defined vector architecture
Expressing an insulin or an insulin analogue through an expression vector in bacteria, wherein the expression vector comprises a first nucleotide sequence encoding a leader peptide of SEQ ID NO:3; a second nucleotide sequence encoding an affinity tag linked to a C-terminal end or a N-terminal end of the leader peptide; a third nucleotide sequence encoding a cleavage site or a Restriction Enzyme (RE) site and ligated to the first nucleotide sequence encoding the leader peptide or to the second nucleotide sequence encoding the affinity tag; and an expression-vector architecture comprising a multiple cloning site (MCS) upstream of the first nucleotide sequence; a ribosome binding site (RBS); a promoter or operator sequence downstream of the RBS; and an antibiotic selection marker upstream of the promoter or operator sequence.
Expression using expression vector comprising SEQ ID NO:1
Expressing the insulin or insulin analogue through an expression vector in bacteria, wherein the expression vector comprises SEQ ID NO: 1.
Overall claim coverage focuses on bacterial expression of insulin or insulin analogues using a vector that encodes a SEQ ID NO:3 leader peptide fused to an affinity tag with a cleavage-site or RE-site ligation arrangement, together with defined upstream and downstream vector elements such as MCS, RBS, promoter or operator, and antibiotic selection marker; additional narrowing is provided by specifying SEQ ID NO:1 and specific bacterial host and component identities.
Stated Advantages
Documented Applications
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