Figure 2.

(a) Coomassie−stained SDS-PAGE gel showing soluble expression of two LC proteins in the Shuffle E. coli strain. Trastuzumab is a kappa LC while LC-B is a lambda LC. (b) Coomassie-stained SDS-PAGE gel showing soluble and insoluble expression of aCD74 LC in the Shuffle strain and strains described in this work, including the lineage preceding the final LC expression strain, SBDG419. A + indicates strains with the indicated genotype and a − denotes strains without. (c) Electron flow diagram in the thioredoxin/glutaredoxin systems responsible for maintaining reduced cytoplasmic cystines in E. coli. Dashed lines represent genetic knockouts responsible for establishing an oxidizing cytoplasm in the Shuffle strain and strain SBDG419. The mutant ahpC* enzyme can reduce glutathione (GSH) and maintains sufficient reducing capacity to preserve the activity of the essential enzyme ribonucleotide reductase (RNR). The trxA knockout was identified in the present work and found to be essential for soluble LC expression in the genetic background of Sutro’s strains. (d) Crude lysate of SBDG419 after expression of Trastuzumab LC in high density fed-batch cell culture and purified LC after a single-step purification using Kappa Select affinity resin.