Evaluation of the purity of recombinant proteins and detection of residual protein contaminants via N-terminal microsequencing and database searching.
The N-terminal amino acid sequences of purified recombinant human gamma-interferon, alpha 2a-interferon and interleukin-2 expressed in E. coli were determined on an Applied Biosystems 477A Protein/Peptide Sequencer and 120A PTH Amino Acid Analyzer. From the raw chromatographic data of these samples, the identity, heterogeneity, amount of methionine-plus species remaining in the final products, and the probable process contaminants were evaluated with the help of computer methods including database searching. General methods to characterize trace contaminants in protein samples were also discussed. Among the sequenced samples, only gamma-interferon was shown to be N-terminal homogeneous. Methionine-containing species were found in interleukin-2 and alpha 2a-interferon. Chicken eggwhite lysozyme was detected in very small amounts in one batch of samples. These results provide valuable information for the development and improvement of preparation methods as well as regulatory responses to recombinant products.
Jin
D Y
DY
National Laboratory of Molecular Biology and Genetic Engineering, Institute of Virology, CAPM, Beijing.
Zhang
Z Q
ZQ
Zhou
Y A
YA
Hou
Y D
YD
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D003553:Cystine / Q000378:metabolism; D016208:Databases, Factual; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*
D016898:Interferon-alpha; D007376:Interleukin-2; D011994:Recombinant Proteins; D003553:Cystine; D007371:Interferon-gamma