TY - JOUR
T1 - Expression of LEKTI domains 6-9′ in the baculovirus expression system
T2 - Recombinant LEKTI domains 6-9′ inhibit trypsin and subtilisin A
AU - Jayakumar, Arumugam
AU - Kang, Ya'an
AU - Mitsudo, Kenji
AU - Henderson, Ying
AU - Frederick, Mitchell J.
AU - Wang, Mary
AU - El-Naggar, Adel K.
AU - Marx, Ute C.
AU - Briggs, Katrina
AU - Clayman, Gary L.
N1 - Funding Information:
This work was supported in part by National Institutes of Health Independent Award R01 DE-13954 (GLC); National Institutes of Health Specialized Program of Research Excellence (SPORE) Grant in Head and Neck Cancer, 1P50-CA-97007; M.D. Anderson Cancer Center Support Grant 5P30 CA 116672; the Alando J. Ballantyne Distinguished Chair; and the Michael A. O’Bannon Endowment for Cancer Research. Dr. Kenji Mitsudo was partly supported by the Nitto Foundation of Nagoya, Japan. We thank Kathryn Carnes, a contract editor for the Department of Scientific Publications at University of Texas, M.D. Anderson Cancer Center, for critical scientific editing of the manuscript, and Mary Wang and Katrina Briggs for technical support.
PY - 2004/5
Y1 - 2004/5
N2 - The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9′ was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9′ (rLEKTI6-9′) with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9′ inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9′ occurred through a noncompetitive mechanism, with inhibitory constants (K i) of 356 ± 12 and 193 ± 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9′ was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9′, recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 ± 10 nM. Taking LEKTI6-9′ as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.
AB - The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9′ was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9′ (rLEKTI6-9′) with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9′ inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9′ occurred through a noncompetitive mechanism, with inhibitory constants (K i) of 356 ± 12 and 193 ± 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9′ was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9′, recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 ± 10 nM. Taking LEKTI6-9′ as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.
KW - Baculovirus
KW - LEKTI
KW - LEKTI domains
KW - Noncompetitive inhibition
KW - SPINK5
KW - Serine proteinases
UR - http://www.scopus.com/inward/record.url?scp=4444223718&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4444223718&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2003.12.004
DO - 10.1016/j.pep.2003.12.004
M3 - Article
C2 - 15039071
AN - SCOPUS:4444223718
SN - 1046-5928
VL - 35
SP - 93
EP - 101
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -