Development of a protein secretion system with the application of sec-dependent protein secretion components.
In order to induce high levels of protein secretion, we have constructed a recombinant plasmid, designated pBP244, into which was incorporated key components of the type-II Sec-dependent secretion system, including LepB (signal peptidase), SecA (ATPase), and SecB (chaperone). The biological activities of the LepB, SecA, and SecB components expressed from genes harbored by pBP244 appeared to play their normal roles. In order to evaluate the protein secretion, a pspA (Streptococcus pneumoniae surface protein A) gene was cloned into pBP244, resulting in pBP438. S. typhimurium harboring pBP438 grown until the stationary phase, secreted a higher level of PspA into the culture supernatants than did the strain harboring pYA3494. The strain harboring pBP438 secreted a supernatant amount 1.71-fold, a periplasmic space amount 1.47-fold, and an outer membrane amount 1.49-fold higher than that of pYA3494. S. typhimurium chi8554 kept the Asd+ plasmid pBP244 and pBP438 for 60 generations in LB broth harboring DAP, thereby indicating that pBP244 and pBP438 were quite stable in the Salmonella strain.
Kim
Sam Woong
SW
Division of Biological Sciences, Pusan National University, Busan 609-735, Korea.
Kim
Young Hee
YH
Yoo
Ah Young
AY
Yu
Jong Earn
JE
Hur
Jin
J
Lee
John Hwa
JH
Cha
Jaeho
J
Kang
Ho Young
HY
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001431:Bacteriological Techniques; D003470:Culture Media / Q000737:chemistry; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000081416:SecA Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003470:Culture Media; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C057842:SecB protein, Bacteria; C051592:pneumococcal surface protein A; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins