The standard 1600+ IMMUNOMETM protein array is designed to enable a comprehensive inspection of the immune system. In this regard, the proteins on this array were selected on the basis of their involvement in the immune response, representing different classes and various types of proteins such as kinases, cytokines, interleukins and transcription factors. Some of the cancer proteins were selected based on “A Census of Human Cancer Genes,” published in 2004 by Michael R. Stratton and co-workers from the Wellcome Trust Sanger Institute in Hinxton, UK.
Other protein arrays utilise peptides, fragment of proteins or purified proteins immobilised on glass slides. Peptides or fragments of proteins only make available a few epitopes per protein and therefore typically miss both discontinuous and numerous linear epitopes, as well as lacking post-translationally modified epitopes. Purified proteins, on the other hand, can be denatured due to aberrant interactions with the array surface, impairing the availability of linear epitopes and disrupting conformational epitopes of the proteins. In this regard, in order to maximise the interaction between autoantibodies and proteins on the array, our KREXTM-based protein array utilises only full-length, native proteins to be immobilised on glass slides where all the epitopes will be present for binding with autoantibodies.
1. Funded Discovery:
Sengenics mainly offers its KREXTM technology in a collaborative, cost sharing model, in which both Parties contribute funds, share IP and share publication authorship. Sengenics shall own the commercialisation rights to any shared IP generated with royalty terms. If you are interested in our Funded Discovery option, leveraging the KREXTM technology, please contact us at [email protected].
2. Licensing Option:
The KREXTM technology may also be accessed through the Sengenics IMMUNOMETM Technology Access Program (iTAP) whereby iTAP partners will be granted a license to fully leverage the KREXTM technology , with open rights to utilise, commercialise and patent discoveries. For more licensing options through iTAP, please contact us at [email protected].
The proteins are immobilised on the glass slide via affinity attachment. Correctly folded, biotinylated proteins are spotted on streptavidin-coated glass slides where the biotin occupies the binding sites on the streptavidin. Affinity capture is a particularly advantageous way to immobilise proteins as the orientation of the immobilised protein is controlled via the folding marker which aids in preserving the structure and function of the arrayed proteins. The folding marker also provides a single point of attachment which negates the problems of protein unfolding, random orientation and non-specific binding that can occur with other protein attachment methods.
It is present at the C-terminal of the protein of interest. However, upon request, it can also be positioned at the N-terminal for certain proteins especially when important/major sites of interactions of the protein are present at the C-terminal.
The folding of the BCCP and its subsequent biotinylation is dependent on the correct folding of the protein from the N-terminal to the C-terminal. This is also the natural direction that mRNA is translated in vivo: 5′ to 3′, N -> C-terminal. The BCCP is positioned at C-terminal so that it can act as a reporter of correct folding of protein, whereby the BCCP will only fold correctly and become biotinylated if the fusion protein if correctly folded from the N-terminal to C-terminal.
The mechanism by which BCCP aids the folding of downstream protein domains is by recruitment of chaperones to the nascent polypeptide and co-overexpression of chaperones or by increasing the overall solubility of the fusion protein.
The attachment of biotin to the lysine residue of the BCCP is catalysed by the enzyme biotin ligase of the host cell in which expression of the BCCP is taking place. Efficient in vivo biotinylation has been observed across different cell types including bacterial, insect and mammalian cells.
E. coli BCCP is an 80-amino acid domain that folds autonomously into a compact, all beta strand structure and is biotinylated post-translationally in vivo on a single, specific lysine residue by the host cell biotin ligase.
Modifications to the protein sequence prior to expression is dependant on the protein structure and localisation. For certain proteins, signal peptides may be added to the existing protein sequence to guide post-translational modification. Also, linkers could be added in between protein of interest and the BCCP folding marker to provide conformational flexibility to the protein.
During translation of large proteins, folding begins in a co-translational manner. Co-translational folding of a fusion partner means that in the majority of cases, the N-terminal domain will start to fold first. If this initial folding results in a properly folded domain, then it is reasonable to assume that the C-terminal domain will also be able to fold correctly. Alternatively, if the N-terminal domain misfolds and starts to present aberrant hydrophobic surfaces, there is a strong likelihood that initiation of the normal folding pathway of the C-terminal domain will be affected, leading to aberrant folding of the C-terminal domain. As we positioned BCCP at C-terminal domain, it would be expected that mis-folding of a N-terminal fusion partner will perturb the proper folding of BCCP and therefore prevent downstream biotinylation of BCCP and immobilisation onto slide.
9 nanolitres of crude protein lysate are spotted in quadruplicate on the array.
One of the major advantages of using the streptavidin-biotin interaction as the basis for array fabrication is that they have very high binding affinity. The high affinity of streptavidin-biotin interaction means that we quickly start to saturate the available biotin-binding sites on the slide surface so a crude normalisation of protein loading can be achieved without pre-adjusting the concentrations of the protein lysates to compensate for differences in the individual expression levels of all 1,600 different proteins.
We use non-contact piezo printing technology to dispense nanolitres of protein onto the array.
No. The addition of biotin to the BCCP acetyl co-A domain permits purification of the proteins by surface capture. The proteins can be simultaneously purified from cellular lysates and immobilised in a single step via the high affinity and specificity exhibited by a streptavidin surface. We term this simultaneous purification and immobilisation as “surface capture”.
16. Since you don’t purify your protein, can we expect competition between endogeneously biotinylated insect cell proteins and biotinylated recombinant proteins for binding site?
Insect cells have five endogenous proteins that have streptavidin binding sites. Under native conditions, we have observed that these proteins do not compete efficiently with biotinylated recombinant proteins for binding to streptavidin. This perhaps reflects low natural expression levels and the fact that in endogenous biotinylated protein, the biotin is typically less solvent accessible in the native protein meaning that the biotin may not be physically accessible to bind to streptavidin.
The proteins on the IMMUNOME protein array are cloned in frame with c-myc tag, which can be used to visualise the proteins on the array (anti-myc assay). To ensure ﬁdelity, clones are sequence-veriﬁed immediately prior to expression in insect cells and western blot analysis is performed after protein expression.
There are over 400 kinases on the IMMUNOMETM platform and to prove the functionality of the proteins, we measure the ability of tyrosine kinases to autophosphorylate in the presence of ATP, which is only possible when the kinases are functional and correctly folded or present in their native conformation. The tumour protein, TP53 present on the array has been proven to be able to bind DNA, which is only possible when the protein is in an active (functional) form.
Yes, homodimeric proteins are expressed autonomously as homodimers, while heterodimeric proteins are expressed as monomers and subsequently co-arrayed to form heterodimers.
Array images can be extracted/analysed by any image quantification software including analysis software provided by scanner manufacturers, third-party software or open source programs. A GenePix Array List (GAL) file for each array is generated to aid image analysis. Please note that GAL files are grid file specific to the Genepix software and may not be compatible with any other software. GAL automatically generates grids on the array slide for auto spot detection supporting image analysis.
The protein array slides can be scanned using any open-format microarray scanners. We would, however, recommend using the Agilent scanner as we’ve found that it is more stable over time than other scanners we have tested.
The array is scanned at 10 µm using any open format scanner.
Depending on the assay type whether it is a biomarker discovery or interaction study, these samples can be run on the IMMUNOME protein array:
Cancer antigens, signalling proteins, transmembrane proteins, kinases and other immunologically relevant proteins. For the full list of proteins, please click here.
The half-life of autoantibodies is 3 weeks. Levels of autoantibodies would be expected to halve in about 20 days. Accumulation of aberrant PTMs will give rise to autoantigens that stimulate re-production of autoantibodies.
Besides Cy-3 and Cy-5, dyes similar to Cy-3 and Cy-5, such as Alexa 647, 555 and 660 dyes, can also be used.
28. Is it possible to include my proteins of interest that are currently not included on the IMMUNOME protein array?
Yes. We can custom produce proteins based on your requirements. Please contact us at [email protected] to discuss this further.
The KREXTM technology is ideally suited for two main applications; autoantibody profiling and interaction profiling. In autoantibody profiling, different applications include biomarker discovery, patient stratification, treatment monitoring, ADR prediction, immunotherapy and irAE prediction. In interaction profiling, we profile interaction of various analytes with the proteins on the array, such as DNA, RNA, protein, peptide, vaccine etc.
30. When a customer finds a biomarker using our platform, the platform is highly sensitive, what other methods can we use to ensure that the biomarker is not just specific to our platform, so therefore could be transferable to a routine clinical setting?
ELISA is the gold standard for the measurement of proteins/immunoassays in a clinical setting . There are also other methods available for results validation such as IP-MS and other MS-based techniques. As ELISAs are potentially less sensitive than protein arrays (although most researchers agree there are no significant differences between biomarkers detected using protein array and ELISA), there is the possibility of not being able to detect low-abundant biomarkers using ELISA.
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