Expression of LEKTI domains 6-9' in the baculovirus expression system: recombinant LEKTI domains 6-9' inhibit trypsin and subtilisin A.
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 (Ki) 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.
Jayakumar
Arumugam
A
Department of Head and Neck Surgery, University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030-4095, USA. ajayakum@mail.mdanderson.org
Kang
Ya'an
Y
Mitsudo
Kenji
K
Henderson
Ying
Y
Frederick
Mitchell J
MJ
Wang
Mary
M
El-Naggar
Adel K
AK
Marx
Ute C
UC
Briggs
Katrina
K
Clayman
Gary L
GL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry; D007313:Insecta; D017434:Protein Structure, Tertiary; D053491:Proteinase Inhibitory Proteins, Secretory; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
D002352:Carrier Proteins; D004220:Disulfides; D053491:Proteinase Inhibitory Proteins, Secretory; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors; D013381:Subtilisins; D014357:Trypsin