Open Access Open Access  Restricted Access Subscription Access

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


Affiliations
1 MI-SWACO, Shafter, CA 93263, United States
2 University of California, San Francisco, San Francisco, CA 94143, United States
3 California State University, Bakersfield, Bakersfield, CA 93311, United States
 

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 (0.75mg/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 (4.67mg/L of media). Enzymatic activity was calculated to be 0.11U/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 15.4mM−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.
User
Notifications
Font Size

Abstract Views: 69

PDF Views: 1




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

Abstract Views: 69  |  PDF Views: 1

Authors

Madison A. Smith
MI-SWACO, Shafter, CA 93263, United States
Jesica Gonzalez
University of California, San Francisco, San Francisco, CA 94143, United States
Anjum Hussain
California State University, Bakersfield, Bakersfield, CA 93311, United States
Rachel N. Oldfield
California State University, Bakersfield, Bakersfield, CA 93311, United States
Kathryn A. Johnston
California State University, Bakersfield, Bakersfield, CA 93311, United States
Karlo M. Lopez
California State University, Bakersfield, Bakersfield, CA 93311, United States

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 (0.75mg/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 (4.67mg/L of media). Enzymatic activity was calculated to be 0.11U/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 15.4mM−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.