Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha.
Successful approaches to protein engineering required that the desired analogs be easily and rapidly obtained in sufficient quantities and purities for unambiguous structural and functional characterizations. Chemical synthesis is the method of choice for engineering small peptides. We now demonstrate that with improved methodologies and instrumentation, total chemical synthesis can be used to produce a small protein in a form suitable for engineering studies. Active human transforming growth factor-alpha (TGF-alpha), a 50 amino acid long protein with three disulfide bonds, has been synthesized and purified in multiple tens of mg amounts in less than 7 days. The purified human TGF-alpha migrated as a single band on SDS-polyacrylamide gels, ran as a single sharp major band at pI = 6.2 on isoelectric focusing gels, displayed an MW = 5546.2 (Th.5546.3) by mass spectrometry, contained three disulfide bonds and had EGF receptor binding, mitogenic and soft agar colony formation activities. The locations of disulfide bonds were found to be analogous to those found in epidermal growth factor (EGF) and in human TGF-alpha expressed in bacteria.
Woo
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Department of Medicine, UCLA School of Medicine 90024-1689.
Clark-Lewis
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Chait
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Kent
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D000595:Amino Acid Sequence; D001322:Autoanalysis; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D010449:Peptide Mapping; D015202:Protein Engineering / Q000379:methods*; D015291:Transforming Growth Factors / Q000302:isolation & purification* / Q000378:metabolism
D004220:Disulfides; D015291:Transforming Growth Factors; D066246:ErbB Receptors