# Improvement of the Transglycosylation Efficiency of a
## 2 Materials and Methods
### Protein Engineering and Recombinant Expression of LnbB in E. coli  
The amino acid sequence for LnbB (GenBank ABZ78855.1) was checked for presence of a signal peptide using the SignalP 5.0 webserver [32] (signal peptide predicted for 1-35). In addition, the sequence was checked for presence of transmembrane helices using the TMHMM 2.0 webserver [33] (C-terminal membrane anchor predicted at 1087-1106). As a result, only the sequence 37-1064 was selected for expression, which is in accordance with the LnbB originally expressed and characterized (35-1064) [3]. The sequence (37-1064) was codon-optimized for _E. coli_ expression and inserted into a pET-24b(+) expression vector by GenScript (Piscataway, NJ, USA) using restriction sites _Ndel_ and _Xhol_ to add a C-terminal His\({}_{6}\)-tag.  
All mutants were constructed using CloneAmp\({}^{\text{TM}}\) polymerase (Takara, Kusatsu, Japan), a set of mutagenic primers listed in Table S1 (Supplementary Materials), and the pET-24b(+)/LnbB (37-1064) plasmid as template. The plasmid template was then digested with Dpnl at 37 \({}^{\circ}\)C overnight, and the PCR products were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Chicago, IL, USA). Competent _E. coli_ DH5\(\alpha\) cells were then transformed with the purified PCR products and plated on LB agar plates supplemented with kanamycin (50 \(\mu\)g/mL). Positive transformants were selected, and corresponding plasmids were extracted using GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA, USA). All constructs were checked by sequencing (Macrogen Europe, Amsterdam, The Netherlands).  
_E. coli_ BL21 (DE3) cells were used for protein production: competent _E. coli_ BL21 (DE3) cells were transformed with the plasmids and selected for kanamycin resistance. Overnight precultures of transformed cells grown at 37 \({}^{\circ}\)C in LB medium containing 50 \(\mu\)g/mL kanamycin were used to inoculate 500 mL LB medium supplemented with 50 \(\mu\)g/mL kanamycin and incubated at 37 \({}^{\circ}\)C. When the OD\({}_{600}\) of each culture reached a value of 0.6-0.8, gene expression was induced using 1 mM IPTG for 20 h at 25 \({}^{\circ}\)C. Cells were harvested by centrifugation (5000\(\times\)\(g\), 15 min), and the pellet was resuspended in binding buffer (20 mM sodium phosphate buffer, 500 mM NaCl, 20 mM imidazole, pH 7.4) before sonication using an UP 400S Ultrasonic processor (Hielscher Ultrasonics, Teltow, Germany). Cell debris was removed by centrifugation (20,000\(\times\)\(g\), 30 min at 4 \({}^{\circ}\)C).  
Proteins were purified on Ni\({}^{2+}\) Sepharose columns (GE Healthcare, Chicago, IL, USA), previously equilibrated with binding buffer. His\({}_{6}\)-tagged proteins were eluted using 250 mM of imidazole in binding buffer. Imidazole was then removed using PD-10 columns (GE Healthcare) according to the supplier's recommendations; the resulting protein buffer was 20 mM sodium phosphate with 100 mM NaCl at pH 7.4. Proteins were visualized on SDS-PAGE (Biorad, Hercules, CA, USA). Protein concentrations were determined at an absorbance of 280 nm using an LnbB extinction coefficient of 183,370 M\({}^{-1}\) cm\({}^{-1}\) and molecular weight of 111,068 g/mol, as predicted by the Expasy web server ([[https://web.expasy.org/protparam/](https://web.expasy.org/protparam/)]([https://web.expasy.org/protparam/](https://web.expasy.org/protparam/)) accessed on 1 November 2021).