Abstract
BackgroundSingle domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. Adding an additional disulfide bond that bridges framework regions is a proven method to increase their melting temperature, however often at the expense of protein production. To fulfill their full potential it is essential to achieve robust protein production of these stable binding elements. In this work, we tested the hypothesis that decreasing the isoelectric point of single domain antibody extra disulfide bond mutants whose production fell due to the incorporation of the extra disulfide bond would lead to recovery of the protein yield, while maintaining the favorable melting temperature and affinity.ResultsIntroduction of negative charges into a disulfide bond mutant of a single domain antibody specific for the L1 antigen of the vaccinia virus led to approximately 3.5-fold increase of protein production to 14 mg/L, while affinity and melting temperature was maintained. In addition, refolding following heat denaturation improved from 15 to 70 %. It also maintained nearly 100 % of its binding function after heating to 85 °C for an hour at 1 mg/mL. Disappointingly, the replacement of neutral or positively charged amino acids with negatively charged ones to lower the isoelectric point of two anti-toxin single domain antibodies stabilized with a second disulfide bond yielded only slight increases in protein production. Nonetheless, for one of these binders the charge change itself stabilized the structure equivalent to disulfide bond addition, thus providing an alternative route to stabilization which is not accompanied by loss in production.ConclusionThe ability to produce high affinity, stable single domain antibodies is critical for their utility. While the addition of a second disulfide bond is a proven method for enhancing stability of single domain antibodies, it frequently comes at the cost of reduced yields. While decreasing the isoelectric point of double disulfide mutants of single domain antibodies may improve protein production, charge addition appears to consistently improve refolding and some charge changes can also improve thermal stability, thus providing a number of benefits making the examination of such mutations worth consideration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0340-3) contains supplementary material, which is available to authorized users.
Highlights
Single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements
This clone produced well in E. coli with typical yields of ~20 mg/L and showed the desired specificity for L1 and high affinity of ~1 nM, it unfolded at 62 °C as determined by circular dichroism (CD) (Table 1), which while fairly typical for Single domain antibodies (sdAb) was insufficient to meet our goal of sdAb that remain stable upon exposure to a sustained temperature of 70 °C
The location of the cysteine substitutions was chosen analogous to the positions reported by Hagihara et al [14], the first group to report on this method of disulfide bond addition in order to increase the melting temperature of a sdAb
Summary
Single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. Single domain antibodies (sdAb), the recombinantly expressed variable region from the unconventional heavy chain only antibodies found in camelids, are renowned for their properties of high affinity coupled with the ability of most to refold into an active form after denaturation [1,2,3,4]. These properties have made sdAb attractive reagents for biotechnology and medical applications, where high affinity and stable reagents are advantageous [5,6,7,8,9]. Our current approach towards engineering the most rugged and robust sdAb recognition reagents is to increase both their melting temperature and solubility [13]
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