Vectors for cloning and expression of proteins, methods and applications thereof
Inventors
Chatterjee, Sohang • Rodrigues, Kavitha Iyer • Ghosh, Maloy • Maity, Sunit • Unnikrishnan, Divya • MUNIRAJU, Yogendra Manjunath Bangalore • Murugesan, Sathyabalan • Mukunda, Pavithra • Prasad, Bhargav • Kamanagowda, Veeresha • Bhattacharjee, Sanghamitra • Dakshinamurthy, Pravin Kumar • Halan, Vivek • Srinivasan, Sankaranarayanan • Hora, Anuradha • Natarajan, Bairavabalakumar • Nair, Karthika • Thanigaivel, Aswini • Maliwalave, Amol • Shenoy, Bharath Ravindra • Rao, Sahana Bhima • Chakrabarty, Subhra Prakash • Dubey, Ashvini Kumar • Khan, Amir • Sharma, Ankurina • Sharma, Rashmi • Tiwari, Anurag • Kumar, Santosh • PATEL, Shivani • M, Nikitha
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11549119-B2
Publication Date
2023-01-10
Expiration Date
Abstract
The present disclosure relates to vectors for cloning and expressing genetic material including but not limiting to antibody gene or parts thereof and methods of generating said vectors. Said vectors express the antibody genes in different formats such as Fab or scFv as a part of intertransfer system, intratransfer system or direct cloning and expression in individual display systems. In particular, phage display technology is used to clone and screen potential antibody genes in phagemid which is followed by the transfer of said genes to yeast vector for further screening and identification of lead molecules against antigens. The present vectors have numerous advantages including uniquely designed inserts/expression cassettes resulting in efficient and smooth transfer of clonal population from phage to yeast vectors resulting in efficient library preparation and identification of lead molecules.
Core Innovation
The document describes engineered vector constructs containing an expression cassette configured to receive and transfer nucleic acids encoding an antibody or a fragment thereof between phagemid and yeast vectors. The cassette includes at least one leader sequence, at least one cloning region for receiving a gene encoding a peptide or a protein that selectively binds to a biologically active ligand, and at least one nucleotide sequence encoding constant region immunoglobulin domains, with the constant region light chain and/or heavy chain comprising at least one change in nucleotide sequence with respect to a corresponding native immunoglobulin constant region.
The expression cassette also includes at least one recombinant tag sequence or a nucleic acid encoding a selection sequence, and provides defined cloning-region architectures using multiple cloning site 1 (MCS I) and multiple cloning site 2 (MCS II). The cloning-region design specifies that MCS I comprises a first restriction site combination in a first sequential order of NdeI, BglII, HindIII, and AscI, and MCS II comprises a second restriction site combination in a second sequential order of NcoI, XbaI, NheI, and NotI.
The document further describes an engineered expression cassette architecture for display of antibody fragments on a surface of a protein expression system, using first and second cloning regions configured to receive genes encoding binding peptides or proteins for biologically active ligands. A display-enabling product is included in the expression cassette, together with enzyme cleavage sites, recombinant tag or selection sequences, and linker sequences, with yeast cell surface display components and an Aga2P display scaffold.
Claims Coverage
The provided claim set includes three independent claims. Across these claims, the inventive coverage is centered on engineered expression-cassette vector constructs with defined cloning regions, constant-region immunoglobulin sequence changes versus native, and configurable tag/selection and display-related cassette elements, with additional coverage for expression cassettes limited to a specified SEQ ID set.
Phagemid-to-yeast transferable vector construct with engineered expression cassette
A vector construct designed to receive a nucleic acid encoding an antibody or a fragment thereof from a phagemid comprising at least one cloning region or from a yeast vector comprising at least one cloning region, or to transfer the nucleic acid encoding an antibody or a fragment thereof to a yeast vector comprising at least one cloning region, wherein the vector construct contains an expression cassette comprising at least one leader sequence; at least one cloning region; at least one nucleotide sequence encoding a constant region immunoglobulin heavy chain or fragment thereof and/or a constant region immunoglobulin light chain or fragment thereof, wherein the constant region light chain or fragment and/or constant region heavy chain or fragment comprises at least one change in nucleotide sequence with respect to a corresponding native immunoglobulin constant region; and at least one recombinant tag sequence or nucleic acid encoding a selection sequence, with the cloning region comprising MCS I and/or MCS II configured with NdeI-BglII-HindIII-AscI for MCS I and NcoI-XbaI-NheI-NotI for MCS II.
Surface display cloning vector with dual cloning regions and defined restriction-site sets
A vector construct designed to clone an antibody or a fragment thereof, containing an expression cassette comprising a promoter sequence; a leader sequence; a nucleotide sequence encoding a product that enables display of a peptide or a protein on a surface of a protein expression system; a first enzyme cleavage site; a first recombinant tag sequence or a nucleic acid encoding a selection sequence; a first linker sequence; a second enzyme cleavage site; a first cloning region operably linked to a second cloning region and a second linker sequence; wherein the cloning regions are configured to receive a gene encoding a peptide or a protein that selectively binds to a biologically active ligand; a second recombinant tag sequence or a nucleic acid encoding a selection sequence; and a terminator sequence; wherein the first and/or second cloning region comprises MCS I and MCS II, with MCS I configured with NdeI-BglII-HindIII-AscI and MCS II configured with NcoI-XbaI-NheI-NotI.
Expression cassette limited to a specified SEQ ID set
An expression cassette having a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, and SEQ ID No. 25.
Overall, the independent claims cover a transferable phagemid/yeast vector construct with an engineered expression cassette that includes leader sequences, defined dual cloning-region MCS I/MCS II restriction-site orders, immunoglobulin constant-region nucleotide changes versus native, and tag/selection sequences; a surface display cloning vector that incorporates display-enabling cassette elements such as enzyme cleavage sites, tag/selection sequences, linkers, and dual cloning regions configured with the same ordered MCS restriction-site sets; and expression cassettes restricted to a defined set of SEQ ID sequences.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?