Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli

HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function.     

Structure and function of an antifreeze polypeptide from ocean pout, Macrozoarces americanus: role of glutamic acid residues in protein stability and antifreeze activity by site-directed mutagenesis.

The successful expression and purification of the recombinant ocean pout antifreeze polypeptide (rAFP) in Escherichia coli have enabled the study of its structure-function relationship by site-directed mutagenesis. The role of carboxyl groups at Glu23 and Glu36 of the rAFP was probed by replacing these residues with either glutamine or alanine residues as both single and double mutants. The AFP mutants were expressed, purified and characterized in terms of primary and secondary structures, thermal stability and antifreeze activities. The properties of these mutants were compared with those of the rAFP. Three distinct functions are identified for the carboxyl groups: (i) the negative charges at positions 23 and 36 are involved in the thermal stability of the polypeptide; (ii) the negative charges at positions 23 and 36 contribute to the thermal hysteretic activities of the polypeptide; and (iii) the negative charge at position 23 and hydrogen-bonding ability at position 36 contribute to the ice-binding activity of the polypeptide.     

Expression of rat alpha-fetoprotein cDNA in Escherichia coli and in yeast

Rat alpha-fetoprotein (AFP) cDNA spanning the complete coding region was cloned and expressed in Escherichia coli as well as in yeast, Saccharomyces cerevisiae. The recombinant AFPs (rAFPs) were purified and characterized. The molecular weights determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were 65,000 for E. coli rAFP and 69,000 for yeast rAFP. Amino acid and N-terminal sequence analyses indicated that yeast cells produced mature AFP by processing the signal peptide properly but E. coli rAFP lacked the N-terminal 53 amino acid residues of preAFP. The yeast rAFP was found to be indistinguishable from authentic AFP in the Ouchterlony immunodiffusion test, radioimmunoassay and estradiol-binding assay while E. coli rAFP was less reactive in these tests. These observations indicated that the rAFP expressed in yeast emulated the properties of authentic AFP.     

The effect of enhanced alpha-helicity on the activity of a winter flounder antifreeze polypeptide.

The antifreeze polypeptide (AFP) from the winter flounder displays partial alpha-helix formation at lower temperatures. To investigate the relationship between antifreeze activity and alpha-helical structure, we designed and then chemically synthesized an AFP analog with enhanced alpha-helicity, and compared its conformation and antifreeze properties with those of the native AFP. The synthetic analog was more helical than the native AFP; however, the antifreeze activity of both peptides were identical. The antifreeze activity of the peptides displayed a strong pH dependence, which paralleled pH-induced changes in helix content. At pH 8.5, the antifreeze activity of both peptides displayed identical concentration dependences. In addition to antifreeze activity measurements, the effects of the peptides on the rate of ice crystal growth were also measured. While both peptides affected the a- and c-axis growth rates of ice crystals, the highly helical analog was able to exert its effect on ice crystal growth rates at 7-8-fold lower concentrations than the native AFP. These data indicate that there is a direct but complex relationship between alpha-helicity and antifreeze activity.     

Structure-function relationship in a winter flounder antifreeze polypeptide. II. Alteration of the component growth rates of ice by synthetic antifreeze polypeptides

Using synthetic analogs of an alpha-helical winter flounder antifreeze polypeptide (AFP) we investigated some important molecular details of the mechanism of action of this AFP. Of the seven peptides synthesized, all but one were amino-terminal deletions of the native AFP. Three of the seven synthetic analogs possessed the same antifreeze activity as the native polypeptide; the other analogs were devoid of antifreeze activity. The growth rates along the a and c axes of ice in solutions of varying concentrations of the three active AFP analogs were examined. The a axis growth rates of ice were inversely proportional to the concentration of the active peptides. The c-axis growth rates of ice were also dependent on peptide concentration. The active peptides enhanced c-axis growth at lower concentrations, while at higher concentrations they inhibited c axis growth. The ability of the peptides to develop antifreeze activity and to alter the a and c axis growth rates of ice depended on the presence of appropriately positioned amino acid residues with hydrogen bonding side chains. From these observations we propose that at low concentrations the AFP, through dipolar interactions and hydrogen bonding, interact with the prism faces of ice retarding a axis growth. At these concentrations, the electrical field of the AFP helix-dipole, like an externally applied field (Bartlett, J.T., van der Heuval, A.P., and Mason, B.J. (1963) Z. Angew. Math. Phys. 14, 599-610), can enhance ice c axis growth. At higher concentrations, the AFP interact with all ice crystal planes and retard both a and c axis growth.     

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.     

High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.     

Structure-function relationships in an antifreeze polypeptide. The role of neutral, polar amino acids.

An alanine-rich, alpha-helical antifreeze polypeptide (AFP) from the winter flounder and seven analogs with variations in the arrangement of neutral, polar amino acids were synthesized. Circular dichroism studies determined that all of the peptides, except for one containing a proline residue, were essentially 100% alpha-helical. Freezing point depression data, analyzed by three methods, showed that rearrangement of polar residues resulted in moderate to complete loss of anti-freeze activity. It was observed that ice crystals grow as hexagonal bipyramids in dilute solutions, with a constant c to alpha axis ratio of about 3.3. Above a critical threshold concentration, which may depend on the AFP to ice binding constant and reflect the onset of cooperative interactions, growth ceases until the temperature is lowered to the freezing point. We conclude that a specific arrangement of both threonine and asparagine (or aspartic acid) residues is critical for maximal activity and that the AFPs probably bind to the pyramidal faces of ice with a specific orientation. These conclusions are consistent with a recent report (Knight, C. A., Cheng, C. C., and DeVries, A. L. (1991) Biophys. J. 59, 409-418) that a similar AFP adsorbs to the [2021] pyramidal planes of ice in dilute solution.     

Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution conformation, and ice growth inhibition studies

A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15N-labeled recombinant polypeptide, show that the polypeptides are alpha-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4 degrees C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins, in which the hydrophobic face is oriented toward the ice at the ice/water interface.     

A hyperactive, Ca2+-dependent antifreeze protein in an Antarctic bacterium

In cold climates, some plants and bacteria that cannot avoid freezing use antifreeze proteins (AFPs) to lessen the destructive effects of ice recrystallization. These AFPs have weak freezing point depression activity, perhaps to avoid sudden, uncontrolled growth of ice. Here, we report on an uncharacteristically powerful bacterial AFP found in an Antarctic strain of the bacterium, Marinomonas primoryensis. It is Ca(2+)-dependent, shows evidence of cooperativity, and can produce over 2 degrees C of freezing point depression. Unlike most AFPs, it does not produce obvious crystal faceting during thermal hysteresis. This AFP might be capable of imparting freezing avoidance to M. primoryensis in ice-covered Antarctic lakes. A hyperactive bacterial AFP has not previously been reported.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

Understanding the mechanism of ice binding by type III antifreeze proteins

Type III antifreeze proteins (AFPs) are present in the body fluids of some polar fishes where they inhibit ice growth at subzero temperatures. Previous studies of the structure of type III AFP by NMR and X-ray identified a remarkably flat surface on the protein containing amino acids that were demonstrated to be important for interaction with ice by mutational studies. It was proposed that this protein surface binds onto the (1 0 [\bar 1] 0) plane of ice with the key amino acids interacting directly with the water molecules in the ice crystal. Here, we show that the mechanism of type III AFP interaction with ice crystals is more complex than that proposed previously. We report a high-resolution X-ray structure of type III AFP refined at 1.15 A resolution with individual anisotropic temperature factors. We report the results of ice-etching experiments that show a broad surface coverage, suggesting that type III AFP binds to a set of planes that are parallel with or inclined at a small angle to the crystallographic c-axis of the ice crystal. Our modelling studies, performed with the refined structure, confirm that type III AFP can make energetically favourable interactions with several ice surfaces.     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

Increased flexibility decreases antifreeze protein activity

Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single α-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg37 HPLC6, nonamidated HPLC6 Ala37, amidated HPLC6 Ala37, and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still α-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface.     

A theoretical model of a plant antifreeze protein from Lolium perenne.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein's inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.     

Artificial multimers of the type III antifreeze protein. Effects on thermal hysteresis and ice crystal morphology

A variant of antifreeze protein (AFP) named RD3 from antarctic eel pout (Lycodichthys dearborni) comprises the type III AFP intramolecular dimer, which is known to exhibit a significant enhancement of thermal hysteresis when compared with the type III AFP monomer (Miura, K., Ohgiya, S., Hoshino, T, Nemoto, N., Suetake, T., Miura, A, Spyracopoulos, L., Kondo, H., and Tsuda, S. (2001) J. Biol. Chem. 276, 1304-1310). Here we genetically synthesized intramolecular dimer, trimer, and tetramer of the type III AFP, for which we utilize the genes encoding the primary sequences of the N-domain, the C-domain, and the 9-residue linker of RD3, and we examined the AFP multimerization effects on thermal hysteresis and ice crystal morphology. Significantly, (i) the thermal hysteresis increases in proportion with the size of the multimers, (ii) a larger size of the multimer exerts the maximum activity at lower concentration, (iii) every multimer changes the morphology of a single ice crystal into a unique shape that is similar but not identical to the ordinary hexagonal bipyramid, and (iv) the size of ice crystal becomes dramatically small with increasing the concentration of the multimer. The thermal hysteresis enhancement of the multimer was detected in both molar and domain bases. These results suggest that a molecule comprising the multiple AFP domains connected in tandem acquires an enhanced affinity for the ice binding.     

Differential scanning calorimetric analysis of antifreeze protein activity in the common mealworm, Tenebrio molitor

Antifreeze proteins (AFP) are able to inhibit the growth of ice-crystals at temperatures below the equilibrium freezing point (Tf) of hemolymph. The analysis of AFP activity has commonly involved the use of direct microscopic observation of a sample following inoculation with ice. The resulting activity, defined as the amount of thermal hysteresis observed between Tf and the subsequent rapid growth of ice, has been reported to range up to 7 degrees C. However, most studies report high level of variation, possibly due to ice-crystal size variability and the presence of non-visible ice nuclei. We describe a new method of analysis of AFP activity using differential scanning calorimetry (DSC). DSC analysis reveals much higher activity, up to 10 degrees C, with less variation observed within a sample, and is not subject to the difficulty of accurate assessment of ice-crystal volume.     

Purification of boiling-soluble antifreeze protein from the legume Ammopiptanthus mongolicus

A boiling-soluble antifreeze protein (AFP) was purified from the winter leaves of Ammopiptanthus mongolicus an evergreen legume species surviving in the cold desert of northwest of China. Purification was achieved by using a procedure consisting of a heat treatment step followed by consecutive chromatography, including ion-exchange chromatography (DEAE-Cellulose 52, Source 15Q), molecular exclusion chromatography with Sephacryl S300, and hydrophobic interaction chromatography (Poros 20HP2). This AFP showed thermal hysteresis activity and could modify the normal growth of ice crystals. The thermal hysteresis activity (THA) of this purified antifreeze protein is 0.15 degrees C at a concentration of 10 mg/mL, and its molecular mass is approximately 28 kD by SDS-PAGE analysis.     

Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast

Certain freeze-intolerant insects produce antifreeze proteins (AFPs) during overwintering including the spruce budworm (Choristoneura fumiferana) and yellow mealworm (Tenebrio molitor) AFP gene families. However, only a few of the isoforms, encoded by their multiple-copy gene families, have been characterized. When expressed in bacterial systems the insect AFPs have to be denatured and refolded in vitro, a procedure that is not uniformly successful, presumably due to the beta-helix structure and the requirement for disulfide bonds. In an attempt to overcome these difficulties, bacterial vectors and hosts that have been developed to produce soluble, folded proteins, as well as a yeast expression system (Pichia pastoris) were employed. Bacterial expression resulted in low quantities of active recombinant protein for certain isoforms. In contrast, both small and large-scale fermentation of recombinant AFP in Pichia yielded substantial protein production (100 mg/L) but functional ice binding activity of protein produced in three different transformed yeast strains (KM71, X33 or GS115) was low. Inappropriate O-linked glycosylation of the Thr-rich AFPs appeared to be partially reversed by mild chemical deglycosylation, but activity remained low. Substantial quantities, as well as activity were recovered when a fish AFP, with disulfide bonds, but without potential Thr glycosylation sites was expressed in the yeast system.     

Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.