Production, characterization, and crystallization of truncated forms of pneumococcal surface protein A from Escherichia coli.
Streptococcus pneumoniae is a major bacterial pathogen that causes diseases such as pneumonia and meningitis in humans. One of the antigens of this organism is pneumococcal surface protein A (PspA). PspA is a virulence factor of the bacteria that has been shown to protect mice against pneumococcal infection. Among several domains of the protein, the amino-terminal part of PspA has been found to be a functional module which is essential for full pneumococcal infectivity. In order to investigate the properties of this protein, several internal fragments of the pspA gene were amplified from S. pneumoniae strain Rxl using the polymerase chain reaction (PCR). The fragments were then cloned and expressed in Escherichia coli in a soluble form using the T7 RNA polymerase pET15b and pET21a vector systems. The size of these fragments ranges from 24 to 32 kDa corresponding to amino acids 67-272 (PspA-206), 1-236 (PspA-236), and 1-272 (PspA-272). The fragments were purified to homogeneity using nickel chelating affinity, size exclusion, and anion-exchange chromatographic methods. During the course of expression of some of the PspA constructs, a shorter fragment was coexpressed due to translational pausing and subsequent secondary translation initiation. Two of the constructs, PspA-206 and PspA-272, were also crystallized allowing for the initiation of a structural elucidation of PspA.
Lamani
E
E
Department of Microbiology, University of Alabama at Birmingham, 933 19th Street South, 545 CHSB-19, Birmingham, Alabama 35294, USA.
McPherson
D T
DT
Hollingshead
S K
SK
Jedrzejas
M J
MJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000235:genetics*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C051592:pneumococcal surface protein A