The experimental results from two rounds of design. (A) Experimental results of single-point mutants. The melting temperatures of mutants are shown as red squares. The affinity of mutants after treatment with 0.3 M NaOH for 24 hours are shown as blue bars. (B) Experimental results of multi-point mutants and single-point mutants. The melting temperatures of mutants are shown as red squares. The affinity of single-point mutants after treatment with 0.5 M NaOH for 24 hours are shown as blue bars, while those of multi-point mutants are shown as green bars. The affinity values were normalized in the graph, with the wild-type EC50 set as 1.

Schematic diagram illustrating the combined effects of single-point mutations. The colors (outer contours representing multi-point mutations and inner solid circles representing the included single-point mutations) and numerical values represent the EC50 values after alkali treatment with 0.5 M NaOH for 24 hours, the EC50 values before alkali treatment, and the Tm values. Blue indicates mutations inferior to the wild type, while red indicates mutations superior to the wild type. The EC50 values before and after alkali treatment are normalized, with the wild type set to 1. (B) Experimental elvalutation of multi-point mutations. Different colors represent the proportions of improvement or decline in Tm, affinity, and alkali resistance of multi-point mutations compared to the corresponding best single-point mutations they include. (C) Distribution of the occurrences of single-point mutations included in the 20 multi-point mutation variants. The length of the bar represents the frequency of occurrence of each mutation.

(A) The SDS-PAGE experimental results depict the proportion of small bands observed after alkali treatment for multi-point mutations and certain single-point mutations exhibiting relatively higher alkali resistance. (B) Structure of the VHH antibody. (C) The multiple sequence alignment of the VHH and several homologous sequences.

(A) The tolerance of acid dissociation and salt dissociation, and the acid resistance of multi-point mutations with strong alkali resistance. All EC50 values are normalized, with the wild type set to 1. (B) Experiment results of residual dynamic binding capacity (DBC) at 10% breakthrough. The bars represent the ratio of the residual DBC after 0.5 M NaOH treatment for 6 hours and 24 hours to the residual DBC before treatment for multi-site mutants and the wild type. (C) Yield in affinity chromatography and the corresponding number of cycles. This figure illustrates the variation in yield during affinity chromatography across multiple cycles.