Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

Abstract

Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM(-1 )cm(-1). No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture.

Figure 1

Schematic of the synthesis of active lysyl oxidase. An inactive propeptide is glycosylated and exported to the extracellular matrix following the incorporation of copper and the formation of the LTQ catalytic cofactor. The propeptide region is cleaved by procollagen C-proteinase at Gly 168 and Asp 169, yielding the propeptide and the 29 kDa active mature enzyme.

Figure 2

Plasmid maps for pLOX06 (Trx), pLOX09 (Nus-A), and pLOX14 (GST). The maps indicate the direction of transcription, the location of the solubility tags, and the insertion points for the wild-type mature LOX gene.

Figure 3

SDS-PAGE gel slices showing overexpressed, solubility-tagged lysyl oxidase. The molecular weight of each solubility-tagged enzyme was determined using the ladder on the left.

Figure 4

Thrombin digest of Nus-A tagged lysyl oxidase. Lane 1: molecular weight marker (MW); Lane 2: undigested tagged enzyme (U); Lane 3: thrombin digested Nus-A tagged LOX (TD).

Figure 5

Phenylhydrazone adduct that is formed when LTQ reacts with phenylhydrazine. Red curve shows the adduct for the Trx tagged enzyme while the green curve shows the adduct for the Nus-A enzyme. The blue trace is that of LOX as purified prior to reacting with phenylhydrazine.