Production and purification of recombinant monoclonal antibodies from human cells based on a primary sequence.

There are challenges to using commercially available antibodies generated in animals, including concerns with reproducibility, high costs, and ethical issues. Here, we present a protocol for generating and purifying recombinant antibodies from human HEK293 suspension culture cells from a primary sequence. We describe the steps to generate antibody heavy and light chain plasmids, followed by transfection of the plasmids into cells and purification of antibodies. This protocol can produce high-yield recombinant monoclonal antibodies at a relatively low cost. For complete details on the use and execution of this protocol, please refer to DeLuca et al. (2021).1.

Generation and diversification of recombinant monoclonal antibodies.

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

The Hec1/Ndc80 tail domain is required for force generation at kinetochores, but is dispensable for kinetochore-microtubule attachment formation and Ska complex recruitment.

The conserved kinetochore-associated NDC80 complex (composed of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including 1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments and 2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to kinetochores and for generation of kinetochore-microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates kinetochore-microtubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex and demonstrate that this region is required for Ska complex recruitment to the NDC80 complex--microtubule interface.

Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore-microtubule dynamics.

Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of kinetochore-microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore-microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.

Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity.

Microtubule (MT) attachment to kinetochores is vitally important for cell division, but how these interactions are controlled by phosphorylation is not well known. We used quantitative approaches in vitro combined with molecular dynamics simulations to examine phosphoregulation of the NDC80 complex, a core kinetochore component. We show that the outputs from multiple phosphorylation events on the unstructured tail of its Hec1 subunit are additively integrated to elicit gradual tuning of NDC80-MT binding both in vitro and in silico. Conformational plasticity of the Hec1 tail enables it to serve as a phosphorylation-controlled rheostat, providing a new paradigm for regulating the affinity of MT binders. We also show that cooperativity of NDC80 interactions is weak and is unaffected by NDC80 phosphorylation. This in vitro finding strongly supports our model that independent molecular binding events to MTs by individual NDC80 complexes, rather than their structured oligomers, regulate the dynamics and stability of kinetochore-MT attachments in dividing cells.

Purification of CREB to apparent homogeneity: removal of truncation products and contaminating nucleic acid.

The cAMP response element binding protein (CREB) is a mammalian transcription factor which regulates the expression of many cellular genes. CREB is commonly expressed in Escherichia coli and purified by heat-extraction followed by affinity chromatography. We have discovered that although this purification yields a reasonably pure product which is active in DNA-binding and functional assays, it contains a large amount of nucleic acid as well as CREB truncation products and other polypeptides. Consequently, this CREB is inadequate for use in biophysical studies including crystallography, and spectroscopic analysis such as analytical ultracentrifugation, FRET, and circular dichroism. We revised the purification protocol to incorporate expression in the Rosetta host strain, nuclease treatment, and denaturing/high salt size-exclusion chromatography. We typically obtain 10mg of CREB per liter of culture media that is 99% homogenous, free of nucleic acid, and amenable to biophysical studies. Comparison of CREB from the original and revised protocols shows similar affinities for the cAMP response element (CRE) but small differences in their secondary structures when assayed by limited proteolysis and circular dichroism.

Molecular characterization of the Tax-containing HTLV-1 enhancer complex reveals a prominent role for CREB phosphorylation in Tax transactivation.

Transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1) is mediated by the viral oncoprotein Tax, which utilizes cellular transcriptional machinery to perform this function. The viral promoter carries three cyclic AMP-response elements (CREs), which are recognized by the cellular transcription factor cAMP-response element-binding protein (CREB). Tax binds to GC-rich sequences that immediately flank the CREs. The coactivator CREB-binding protein (CBP)/p300 binds to this promoter-bound ternary complex, which promotes the initiation of HTLV-1 transcription. Protein kinase A phosphorylation of CREB at serine 133 facilitates transcription from cellular CREs by recruiting CBP/p300 via its KIX domain. However, it remains controversial whether CREB phosphorylation plays a role in Tax transactivation. In this study, we biochemically characterized the quaternary complex formed by Tax, CREB, KIX, and the viral CRE by examining the individual molecular interactions that contribute to Tax stabilization in the complex. Our data show KIX, Ser(133)-phosphorylated CREB, and vCRE DNA are all required for stable Tax incorporation into the complex in vitro. Consonant with a fundamental role for CREB phosphorylation in Tax recruitment to the complex, we found that CREB is highly phosphorylated in a panel of HTLV-1-infected human T-cell lines. Significantly, we show that Tax is directly responsible for promoting elevated levels of CREB phosphorylation. Together, these data support a model in which Tax promotes CREB phosphorylation in vivo to ensure availability for Tax transactivation. Because pCREB has been implicated in leukemogenesis, enhancement of CREB phosphorylation by the virus may play a role in the etiology of adult T-cell leukemia.