Hyung-Kwon Lim

Improvement of heterologous protein productivity by controlling postinduction specific growth rate in recombinant Escherichia coli under control of the PL promoter

To develop the optimal operational strategy for the PL promoter of interferon (IFN)‐γ‐producing Escherichia coli, some rational medium feeding methods, carried out just after temperature induction to control the postinduction specific growth rate (μi), were designed. Using experimental data from various batch cultures, we correlated μi with specific IFN‐γ production rate (qpi) to find out how μi just after induction affected qpi. It was revealed that sustaining μi above a certain value after induction was the key factor for high‐level production of IFN‐γ. Stepwise and constant‐rate medium feeding, in which the temperature induction was done simultaneously, at the late‐exponential growth phase of batch culture resulted in a dramatic extension of the production period as a result of sustaining μi after temperature induction. These methods led to the overcoming of harsh conditions after temperature induction and the improvement of IFN‐γ productivity. Finally, by both a high‐cell‐density culture using an exponential fed‐batch culture for cell growth and constant‐rate medium feeding after temperature induction for IFN‐γ production, we accomplished an increase in IFN‐γ production by 23‐fold, 7.43 g/L, as compared with that of batch culture.

Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter.

Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.

Effects of Trx2p and Sec23p expression on stable production of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae

The S domain of hepatitis B virus surface antigen (sHBsAg) is the primary component for vaccine development against virus infection. For stable expression of sHBsAg in recombinant Saccharomyces cerevisiae, new accessory genes necessary for foreign protein expression were screened by DNA microarray. Among 600 genes of interest, genes coding for an activating protein of ATPase in Hsp90 (Aha1p), S. cerevisiae DnaJ (Scj1p), thioredoxin 2 (Trx2p) and a GTPase-activator specific for Sar1 (Sec23p) as well as Pdi1p were selected in transcriptome analysis, which are known to facilitate disulfide bond formation or induce protein transport in the secretion pathway. Individual and combinatorial expression of SEC23, TRX2 and PDI1 increased total sHBsAg concentration by 1.9-6.5-fold, relative to the control strain expressing sHBsAg only. Additionally, moderate expression of Kex2p protease able to cut off the signal peptide enhanced the portion of the authentic sHBsAg to total sHBsAg. Fed-batch fermentation of the S. cerevisiae 2805 strain coexpressing the sHBsAg, SEC23, PDI1 and KEX2 genes resulted in 70.6mg/L final sHBsAg concentration which was 5.6 times higher than that of the control. Transmission electron microscopic analysis of the yeast cells elucidated the effects of the accessory gene coexpression on the intracellular localization of sHBsAg. Like PDI1, coexpression of both SEC23 and/or TRX2 newly isolated in this study is expected to improve the target protein expression in S. cerevisiae.