Protein molecular weight and pI, tag on or off

Worked example

• GFP (P42212) behind a His6 tag and a TEV site, 258 residues as expressed: 29154.69 Da, pI 5.94, by ProtParam's residue masses and the Bjellqvist pK set as ProtParam applies it.
• The His6 tag and the TEV site (ENLYFQ|G/S) are found by their motifs; the cut TEV makes leaves a 239-residue product of 26943.37 Da, pI 5.67, once you confirm it.
• ε280 is 23,505 M⁻¹cm⁻¹ as expressed and 22,015 after the cut: the tag has no Trp or Tyr, so the mass changes and the absorbance does not.

Method

MW = Σ residue masses + H₂O; pI by bisection on the net charge with the Bjellqvist pK set as ProtParam applies it

• Average molecular weight from the sequence. ExPASy ProtParam (Gasteiger et al. 2005); the fixtures are ProtParam's printed weights, retrieved 5 October 2026.Checked against Expasy ProtParam within 0.01 Da on 22 sequences with ProtParam's residue masses (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.
• Theoretical isoelectric point. Bjellqvist B et al. Electrophoresis 1993;14:1023-1031; 1994;15:529-539; as ExPASy ProtParam applies them.Checked against Expasy ProtParam within 0.01 on 22 sequences, never a bound of the pH 0 to 14 search (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.
• Molar extinction coefficient at 280 nm. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995;4:2411-2423 (read 5 October 2026).Checked against Expasy ProtParam exactly on 22 sequences in both cysteine conventions (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.
• Tags and protease sites by motif. Peptide tag and protease recognition motifs from the cited papers; a cut is proposed at the site nearest an end and confirmed by the person, never inferred from the fold.Unit-tested on the fixture construct: the His6-TEV construct's cleavage product is the untagged fixture sequence (6 October 2026).
• Peptide as SMILES. A linear peptide of L-amino acids with free amine and acid ends, neutral, cysteines as free thiols, drawn as RDKit's MolFromSequence draws it (Daylight SMILES; RDKit 2026.03.6 as the oracle).Checked against RDKit 2026.03.6 MolFromSequence: the same canonical SMILES on 89 peptides and the 12 refusals exact (6 October 2026).