Community-Wide Experimental Evaluation of the PROSS Stability-Design Method

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields

“…A10 is an anti-Her2 nanobody isolated by panning a synthetic library in which the clones only differ in the CDR positions [ 4 ] and was previously selected as a model for benchmarking the performance of the PROSS server [ 13 ]. While only mutations in the framework were assessed to preserve the CDR-based paratope in the benchmark work, here, we employed PROSS to identify fitter mutants by considering the whole VHH sequence.…”

“…Overall, two submissions of the A10 model were made to the PROSS server. The first only enabled mutations at framework residues (positions 1–29, 36–54, 62–100 and 110–122, described in [ 13 ]), and the third submission allowed the design of the complete nanobody, including the CDR amino acids. The results of the latter served as a reference for selecting the variants to experimentally express and analyze ( Table 1 ).…”

“…There are several examples demonstrating the feasibility of this approach, but these are still the results of case-by-case projects that require a major input from specialized researchers and often require access to structural information [ 9 , 10 , 11 , 12 ]. On the other hand, we recently confirmed that publicly accessible algorithms that require minimal data inputs can rapidly and effectively help in the identification of protein variants that exhibit astonishingly higher yields than the original sequences [ 13 ]. A major challenge for stability design methods is the preservation of the protein function despite the introduction of stabilizing mutations.…”

“…Technically, this is simple to obtain because the PROSS interface enables the specification of selected amino acids that the algorithm will not propose mutations for. Under these conditions, the program proposed a set of point mutations in the nanobody framework, and the corresponding variants were expressed and evaluated, and the results were discussed in a recent paper [ 13 ]. Here, we further assessed the capacity of the PROSS to suggest fitter mutants by removing the protection for the residues present in the CDRs.…”

Obituary: Dan S Tawfik (1955–2021)

“…Together with Danny, we have developed general and automated methods that we and others have used to design enzymes, binders, and vaccine immunogens with superior stability [17], expression yields [26,27], and activity profiles [28]. This strategy builds directly on Danny's insights on the stability‐threshold effect and the potential to improve promiscuous enzyme activities [13,24] and has been used by researchers around the world [29]. It is a shining testament to Danny's versatility and open‐mindedness that he enthusiastically supported and participated in this line of computational research which differed from his main interests.…”