Recombinant CD74 polypeptides
Inventors
Meza-Romero, Roberto • Benedek, Gil • Vandenbark, Arthur A.
Assignees
Oregon Health and Science University • US Department of Veterans Affairs
Publication Number
US-10689446-B2
Publication Date
2020-06-23
Expiration Date
2037-06-21
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Abstract
Disclosed are recombinant CD74 polypeptides mutated relative to the naturally occurring CD74 polypeptides with improved properties such as binding of CD74 ligands such as MIF and RTL1000 as well as polynucleotides that encode the polypeptides, expression vectors comprising the polynucleotides, bacteria that include the expression vectors, and methods of making the recombinant polypeptides.
Core Innovation
The invention relates to recombinant CD74 polypeptides mutated relative to naturally occurring CD74 polypeptides with improved properties such as enhanced binding to CD74 ligands including macrophage migration inhibitory factor (MIF) and RTL1000. The disclosure includes polynucleotides encoding these polypeptides, expression vectors, bacterial cells harboring these vectors, and methods for producing the recombinant polypeptides. Specifically, novel soluble recombinant forms of CD74 that bind natural and recombinant CD74 ligands are provided, which were not commercially available at the time of filing.
The problem addressed is the unavailability of active soluble recombinant CD74 polypeptides capable of binding to known CD74 ligands such as MIF, D-DT, RTL1000, and DRα1. Such active forms are needed to facilitate further research. The invention overcomes issues related to dimerization of CD74 trimers during purification and storage by engineering mutations like C27S, resulting in soluble forms that retain binding activity and can be produced in bacterial cells with codon-optimized nucleotide sequences.
Claims Coverage
The patent contains two independent claims focusing on recombinant CD74 polypeptides and their compositions, detailing specific sequences and modifications.
Recombinant polypeptide composition
A recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
Polypeptide comprising a label
The polypeptide of SEQ ID NO: 1 or SEQ ID NO: 3 further comprising a label selected from fluorescent dye, enzyme, protein tag, fluorescent protein, or biotin.
The independent claims cover recombinant polypeptides of specific CD74 sequences (SEQ ID NO: 1 or 3) and their labeled forms, encompassing particular mutations and tags that improve utility and binding properties.
Stated Advantages
Provides active soluble recombinant CD74 polypeptides capable of binding natural and recombinant ligands such as MIF, D-DT, RTL1000, and DRα1.
Enables production of soluble CD74 forms that avoid dimerization issues, resulting in more accurate binding data in vitro.
Allows for the recombinant production of CD74 in bacterial cells using codon-optimized expression constructs, facilitating efficient protein preparation and purification.
Documented Applications
Use of recombinant soluble CD74 polypeptides to facilitate research by binding to natural and recombinant CD74 ligands including MIF, D-DT, RTL1000, and DRα1.
Purification and detection of CD74 polypeptides via incorporation of labels such as fluorescent dyes, enzymatic tags, protein tags, or biotin for research and analytical purposes.
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