Surface supercharged human enteropeptidase light chain shows improved solubility and refolding yield

Abstract

Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Specifically, protein surface supercharging (N6D, G21D, G22D, N141D, K209E) of the protein increased the solubility more than 100-fold. Replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

“…Aggregation resistance has been increased through the method of supercharging proteins by engineering in an excess number of acidic [25,45,85] or basic residues [58] or alternatively protein net charge can be increased by covalently attaching charged amino acid tags [83,87]. Similarly, heat resistant antibody V H domains isolated from a combinatorial library of mutations generated by phage display generally had a disproportionate number of acidic groups [3,26,39].…”

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

“…This process enhances solubility and causes intermolecular repulsion even in the unfolded state, which in turn prevents aggregation [17]. Fusion to supercharged polypeptides enhances protein delivery in vitro and in vivo in a process that is clathrin-and energydependent, involving binding to anionic cell surface proteoglycans and endocytosis [2].…”

Human enteropeptidase light chain: Bioengineering of recombinants and kinetic investigations of structure and function

“…Assays during purification were in the absence of CaCl 2 or Triton X-100, which were subsequently found to enhance the activity of Y174R consistent with a report on solubility problems with recombinant hEP. 30 Subsequent kinetic analyses including 0.02 mM CaCl 2 and 0.01% Triton X-100 show that both variants have similar K M and k cat values for Z-Lys-SBzl (Table I), suggesting that Y174R is less soluble than R96Q. The single-step STI affinity purification yielded highly purified enzymes [ Fig.…”

“…29 Others created, in E. coli, a Y174R variant of bEP L that showed an approximate four-fold improvement in specificity toward the GD 4 K$ IVGG substrate as a result of the proposed salt bridges by R174 with Asp-P3 and Asp-P4. 22 Recently, a supercharged rhEP L (rhEP L sc) was created in E. coli to improve enzyme solubility and refolding yields 30,31 and an x-ray crystal structure of this mutant was obtained (PDB code: 4DGJ). 28,30,31 In an effort to improve the utility of rhEP L for processing fusion proteins and to better understand the structure and function of hEP L , two rhEP L variants were created and produced as active enzymes secreted by P. pastoris.…”

“…22 Recently, a supercharged rhEP L (rhEP L sc) was created in E. coli to improve enzyme solubility and refolding yields 30,31 and an x-ray crystal structure of this mutant was obtained (PDB code: 4DGJ). 28,30,31 In an effort to improve the utility of rhEP L for processing fusion proteins and to better understand the structure and function of hEP L , two rhEP L variants were created and produced as active enzymes secreted by P. pastoris. Kinetic analyses employed synthetic D 4 K and D 4 R p-nitroanilde (D 4 K-pNA and D 4 R-pNA) substrates and the N-a-carbobenzyloxy-Llysine-thiobenzyl ester (Z-Lys-SBzl) substrate for comparison with other reports.…”