Production of Single-Domain Antibodies in Pichia pastoris

Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogenic contamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.