Biologic and biochemic properties of recombinant platelet factor 4 demonstrate identity with the native protein.
Platelet factor 4 (PF4) is a 70 amino acid protein released from the alpha-granules of platelets after activation. The exact biologic function of this protein is unknown. We have constructed an expression vector for recombinant PF4 (rPF4) in the T7-based promoter vector pT7-7 to better study the relationship between PF4 structure and function. The protein was expressed in Escherichia coli and purified to homogeneity by heparin-agarose affinity chromatography and reverse-phase high-performance liquid chromatography. Purity of protein was confirmed by immunoblot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which resulted in a single component with a molecular weight of 8,000 daltons. The amino acid composition and sequence of the N-terminal 20 residues showed that rPF4 is identical to PF4 prepared from human platelets (hPF4), except for an N-terminal initiating methionine residue. Immunoblots revealed that rPF4 and hPF4 bound polyclonal anti-hPF4 equally well, while chemotaxis experiments demonstrated similar potencies as neutrophil attractants. Dose-dependent neutrophil chemotactic responses and competitive studies with polyclonal anti-hPF4 antiserum further demonstrate similar chemotactic properties of the two PF4 species. In conclusion, our data show that this recombinant protein and the native protein appears to have similar immunologic, heparin-binding, and chemotactic properties. The chemotactic properties of hPF4 appear to be entirely intrinsic to the protein and not due, in part, to any contaminating protein. Furthermore, our expression vector should prove useful for the construction of recombinant forms of PF4 to investigate structure/function relationships of this biologically important protein.
Park
K S
KS
Division of Hematology, Children's Hospital of Philadelphia, PA 19104.
Rifat
S
S
Eck
H
H
Adachi
K
K
Surrey
S
S
Poncz
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002633:Chemotaxis / Q000276:immunology / Q000502:physiology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors / Q000502:physiology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology; D010957:Plasmids; D010978:Platelet Factor 4 / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*
D000596:Amino Acids; D011994:Recombinant Proteins; D010978:Platelet Factor 4