Cold Adaptation, Ca²⁺ Dependency and Autolytic Stability Are Related Features in a Highly Active Cold-Adapted Trypsin Resistant to Autoproteolysis Engineered for Biotechnological Applications

<div><p>Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca²⁺ dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10^° C and at least 50% more active than pig trypsin up to 50^° C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca²⁺ concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca²⁺ dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.</p> </div

Unrooted phylogenetic tree of 28 trypsins inferred from their amino acid sequence alignment.

<p>Five phylogenetically-related clusters were inferred from this analysis: cluster A of shrimp and prawn trypsins (both cryophilic and mesophilic), cluster B of crayfish trypsins, cluster C of other non-crustacean Arthropoda, cluster D of cryophilic vertebrate trypsins, and cluster E of trypsins from mesophilic vertebrates. Cluster A trypsin sequences are shown to be very closely related to krill trypsins KT1 and KT4.</p

Trypsin-like activity versus Ca²⁺/enzyme molar ratio for KT1 and PT.

<p>A comparison plot between KT1 activity and PT activity at equivalent calcium/enzyme molar ratios (referred to on the right vertical axis) have been constructed to compare both calcium dependencies and extract more detailed information about the activity differences at low and high calcium concentrations.</p

Wall-eye stereogram representation of the main features of the KT1 molecular model.

<p>The 3D molecular model of KT1 is represented in a wall-eye stereo view. β-sheets are represented in purple, α-helixes in cyan and loops in pink. The solvent accessible molecular surface is shown as a transparent gray envelope. Particular features of KT1 are highlighted. The TAL is showed in green and the CBL in yellow. The chelated Ca²⁺ atom is displayed as an orange sphere. Active site extending loops are shown in violet in the right-lower corner. The three active residues are represented at the middle-right of the figure. At the top, three of the putative cleavage sites amino acid residues are shown: K163, K197 and R230. The primary K96 cleavage site is shown at the bottom of the figure.</p

SDS-PAGE and zymographic analysis of KT1 mutants.

<p>(A) SDS-PAGE of the undigested and digested wild type KT1 and K96H and K96H+R230E mutants. Lane 1: purified K96H mutant. Lane 2: digested K96H mutant. Lane 3: purified K96H+R230E mutant. Lane 4: digested K96H+R230E mutant. Lane 5: purified wild type KT1. Lane 6: digested wild type KT1. Lane 7: PT used for digestion (1×10^-1 U/ml). (B) SDS-PAGE of the digestion of the K96S mutant with different amounts of PT for digestion. Lane 1: no PT added. Lane 2: 8×10^-4 U PT/ml. Lane 3: 4×10^-3 U PT/ml. Lane 4: 2×10^-2 U PT/ml. Lane 5: 1×10^-1 U PT/ml. (C) Zymograms of the corresponding digestion of mutant samples in (B) compared to the digestion of wild type KT1 under the same conditions. Zymograms are presented as negative images for better clarity.</p

pH dependency of the catalytic activity of the purified krill trypsin.

<p>Comparative plots of enzymatic activity versus pH for the isolated cryophilic krill trypsin and the mesophilic pig trypsin.</p

Wall-eye stereograms of the structural comparison between calcium binding loops of KT1, PT and CFT.

<p>Wall-stereo view of the molecular superposition of the calcium binding loops (yellow), trailing activation loops (green) and surrounding residues (violet) in the cryophilic krill trypsin 1 (KT1), the mesophilic pig trypsin (PT), and the homologous crayfish trypsin (CFT), showing the structural differences discussed in the text. CBLs and TALs are viewed from a point where the line of view is approximately perpendicular to the molecular surface of the protein. In this way, the closest residues to the viewer are the surface residues, and receding side chains are buried inside the protein structure. Amino acid side chains mentioned in the text are labeled. H80 hydrogen bonds in KT1 are shown as cyan dotted lines.</p

Temperature dependency of the catalytic activity of the purified krill trypsin.

<p>Comparative plots of enzymatic activity versus temperature in the range from 4 to 80^°C for the isolated cryophilic krill trypsin and the mesophilic pig trypsin on two substrates, casein (A) and BAPNA (B). The corresponding Arrhenius plots are also shown, including the corresponding linear regressions at each side of the maximum.</p

SDS-PAGE and Western blot analysis of the recombinant purified proteins and their post-activation products.

<p>(A) SDS-PAGE of the products of the <i>E</i>. <i>coli</i> BL21(DE3)/pET22b-KT1 expression system. Lane 1: purified inactive proteins from the soluble cytoplasmic cell fraction. Lane 2: end products after pig trypsin activation of the sample in lane 1. (B) Western blot of polyhistidine-tagged proteins present in the corresponding samples in (A).</p

SDS-PAGE and zymographic analysis of the recombinant trypsinogen and its exogenous activation to produce active KT1.

<p>(A) SDS-PAGE of the products of the <i>E</i>. <i>coli</i> TB1/pMALc2E-KT1 expression system. Lane 1: purified inactive proteins from the soluble cytoplasmic cell fraction. Lane 2: purified proteins digested with enterokinase. Lane 3: purified expression control of uninduced cells. (B) SDS-PAGE of the products of the <i>E</i>. <i>coli</i> BL21(DE3)/pET22b-KT1 expression system. Lane 1: purified inactive proteins from the soluble cytoplasmic cell fraction. Lane 2: purified expression control of uninduced cells. (C) SDS-PAGE of digestions of the purified products of the <i>E</i>. <i>coli</i> BL21(DE3)/pET22b-KT1 expression system with different amounts of pig trypsin for the same incubation time. Lane 1: control with 5×10^-1 U/ml of pig trypsin with no substrate. Lane 2: 5×10^-1 U/ml. Lane 3: 1×10^-1 U/ml. Lane 4: 2×10^-2 U/ml. Lane 5: 4×10^-3 U/ml. Lane 6: 8×10^-4 U/ml. Lane 7: 1.6×10^-4 U/ml. Lane 8: undigested control of the original sample with no pig trypsin. (D) Zymograms of the corresponding samples in (C). The control includes pig trypsin at the same amounts with no recombinant trypsinogen. Zymograms are presented as negative images for better clarity.</p