Engineering Tissue Inhibitors of Metalloproteinases Using Yeast Surface Display

Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display

“…To further investigate the MMP‐3cd inhibition of minimal TIMP variants, enzyme inhibition assays were performed using soluble active MMP‐3cd protein soluble minimal TIMP variant peptides, and soluble N‐TIMP‐1 protein as reference. The tight inhibition binding assays were performed using an MMP cleavable fluorogenic substrate (Raeeszadeh‐Sarmazdeh et al, 2022; Raeeszadeh‐Sarmazdeh, Greene, et al, 2019; Toumaian & Raeeszadeh‐Sarmazdeh, 2022). The minimal TIMP variants, mTC1, mTC2, and mTC3 showed the inhibition constants in the picomolar range (323–568 pM) with mTC1 having the lowest K i and mTC3 the highest value compared to N‐TIMP (385 pM) which were consistent with the data previously reported for TIMPs (Raeeszadeh‐Sarmazdeh et al, 2022) and yeast surface display binding data (Figure 4b).…”

“…Binding between bMMP‐3 and N‐TIMP‐1 or minimal TIMP variants was performed on the yeast surface similar to protocols done previously (Raeeszadeh‐Sarmazdeh et al, 2022; Toumaian & Raeeszadeh‐Sarmazdeh, 2022). The yeast cells displaying N‐TIMP‐1 or minimal TIMP variants were grown and induced as previously described in SD‐CAA (pH 6) media at 30°C shaker and induced in SG‐CAA media at 30°C for 20 h. The induced yeast cells were pelleted at OD 600 of 0.4 by centrifuge at 5000 Xg for 4 min at 4°C and washed three times with PBSA buffer.…”

“…The cut vector and shuffled TIMPs PCR products were concentrated using PelletPaint per manufacturer's instructions. The double‐digested pCHA vector and final PCR product were electrotransformed into EBY100 yeast cells as previously described (Raeeszadeh‐Sarmazdeh et al, 2022; Raeeszadeh‐Sarmazdeh, Greene, et al, 2019; Toumaian & Raeeszadeh‐Sarmazdeh, 2022). The minimal TIMP library was then harvested and resuspended in SDCAA (pH 4.5) media.…”

“…MMP‐3cd and MMP‐9cd proteins were expressed, solubilized, purified, refolded, and activated as previously described (Bolt et al, 2022; Raeeszadeh‐Sarmazdeh et al, 2022; Toumaian & Raeeszadeh‐Sarmazdeh, 2022). Briefly, pET‐pro‐MMP‐3cd or pro‐MMP‐9cd were expressed in pLysRosetta Escherichia coli cells by induction with isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) at a final concentration of 0.5 μM for 3–4 h at 37°C shaker.…”

“…MMP‐3cd and MMP‐9cd activation was performed using 4‐aminophenylmercuric acetate (APMA). Recombinant N‐TIMP‐1 protein was expressed in pTT mammalian expression vector and expressed in FreeStyle™ 293‐F cells (ThermoFisher), extracted and concentrated from the media, and purified using ionic exchange and gel extraction chromatography as previously described (Bolt et al, 2022; Raeeszadeh‐Sarmazdeh et al, 2022; Toumaian & Raeeszadeh‐Sarmazdeh, 2022).…”