Purified protein samples were buffer-exchanged into 0.1% acetic acid using a 10k MWCO Vivaspin (Sartorius) and diluted to a final concentration of 0.5 mg ml -1 . Mass spectrometry was performed using a 1200 series Agilent LC, 5 µL injection into 5% acetonitrile (with 0.1% formic acid) and desalted inline for 1 min. Protein was eluted over 1 min using 95% acetonitrile with 5% water. The resulting multiply charged spectrum was analysed using an Agilent QTOF 6510 and deconvoluted using Agilent MassHunter Software. Library construction. Saturation mutagenesis. Positions were individually randomized using NNK codon degeneracy. DNA libraries were constructed by overlap extension PCR. The linear library fragments and the pET29b(+) vector were digested using NdeI and XhoI endonucleases, gel-purified and subsequently ligated using T4 DNA ligase.